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IMMUNITY 

PROTECTIVE  INOCULATIONS  I 
INFECTIOUS  DISEASES 

AND 

SERUM-THERAPY 


GEORGE  M.  STERNBEEG,  M.D.,  LL.D. 

SURGEON-GENERAL    U.    S.   ARMY 

EX-PRESIDENT  AMERICAN  PITBLIC  HEALTH  ASSOCIATION  ;  HONORARY   MEMBER  OF  THE 

EPIDEMIOLOGICAL  SOCIETY  OF  LONDON,    OF  THE   ROYAL  ACADEMY  OF  MEDICINE 

OF    ROME.    OF    THE    ACADEMY  .OP    MEDICINE    OF    RIO    DE    JANEIRO, 

OF  THE  SOCIETE  FRAN^AISE  D'HYGIENE,   ETC.,   ETC. 


NEW   YORK 
WILLIAM  WOOD  AND  COMPANY 

1895 


Copyright.  1895.  by 
WILLIAM  WOOD  AND  COMPANY 


TROW   DIREOTOnv 

PRINTINQ  AND   BOOKB'NDING  COMPANY 

N&W  YORK 


Bionefieal 
ISkmj 

aw 

%\S' 

PEEFAOE. 

The  following  quotations  from  a  paper,  entitled  "  Prac- 
tical Results  of  Bacteriolog-ical  Researclies,"  read  by  the 
writer  at  the  meeting  of  the  Association  of  American 
Physicians,  May  24,  1892,  may  serve  as  a  preface  to  the 
present  volume : 

"  Science  does  not  demand  practical  results,  but  in- 
vestigates for  the  purpose  of  establishing  facts  and  ex- 
plaining phenomena.  And  bacteriology,  as  a  branch  of 
natural  history,  is  entitled  to  equal  consideration  with 
other  departments  of  scientific  research.  Indeed,  the 
low  organisms  known  as  bacteria  offer  many  advantages 
"^or  the  study  of  fundamental  biological  problems,  and 

^rom  a  scientific  point  of  view. 

"  But  medicine  is  eminently  practical  in  its  aims,  and 
practising  physicians,  as  well  as  intelligent  laymen,  are 
"rapt  to  meet  every  announcement  of  a  new  discovery  in 
pathology  with  the  question,  '  Does  it  aid  in  the  cure  of 
disease  ?  '  Heretofore  the  bacteriologist  has  been  com- 
pelled to  admit  that  the  demonstration  of  the  specific 
cause  in  a  considerable  number  of  infectious  diseases, 
which  has  been  obtained  through  his  researclies,  has  not 
resulted  in  the  discovery  of  a  specific  treatment  for  these 
diseases.     At  the  present  moment  we  are  in  possession 

Ispf  experimental  data  which  open  up  to  us  a  vista  of  pos- 

\sibilities  in  specific  treatment  unsuspected  a  year  or  two 

"vbago. 

624414 


v^the  researclies  already  made  have  borne  abundant  fruit 


IV  PREFACE. 

"  The  researches  of  bacteriologists  have  established  the 
fact  that  the  pathog-enic  action  of  those  bacteria  which 
have  been  shown  to  be  concerned  in  the  etiology  of  spe- 
cific infectious  diseases  is  due  to  the  formation  of  toxic 
products  during  the  active  development  of  the  bacterial 
cells.  The  discovery  of  these  toxins  and  toxalbumins 
has  led  to  a  line  of  research  work,  the  object  of  which  is 
to  isolate  and  study  each  of  these  toxic  products  separ- 
ately by  the  methods  of  chemistry  and  of  experimental 
pathology. 

"  In  the  course  of  these  experiments,  and  of  the  ex- 
tended researches  -(vhich  have  been  made  with  reference 
to  the  explanation  of  natural  and  acquired  immunity,  the 
remarkable  discoveries  which  will  occuj^y  our  special  at- 
tention have  been  made.     .     .     . 

"  Evidently  the  production  of  an  antitoxin  during  an 
attack  of  any  one  of  the  infectious  diseases  would  ac- 
count for  recovery  in  non-fatal  cases  ;  and  it  may  be  that 
this  is  the  true  explanation  of  self -limitation  in  diseases 
of  this  class.  If  Nature  adopts  this  method  of  cure,  we 
but  follow  her  if  we  seek  to  introduce  more  of  the  anti- 
toxin for  the  purpose  of  arresting  the  progress  of  cases 
of  unusual  severity  and  fatal  tendency.     .     .     . 

"  Although  the  production  of  these  antitoxins  in  con- 
siderable amounts  for  therapeutic  use  will  be  attended 
with  difficulties,  there  can  be  no  doubt  that  methods  will 
be  devised  for  obtaining  them  on  a  large  scale  as  soon  as 
it  is  definitely  established  that  they  may  be  successfully 
used  as  specifics  in  the  treatment  of  infectious  diseases. 

"  In  diseases  which  are  common  to  man  and  the  lower 
animals  the  source  from  which  they  maj"  be  obtained  is 
evident ;  but  in  diseases  peculiar  to  man  we  do  not  at 
present  see  just  how  thej^  are  to  be  obtained.    Reasoning 


PREFACE.  V 

from  the  analogy  afforded  by  the  experimental  evidence 
heretofore  referred  to,  we  infer  that  the  blood  and  tissue 
juices  of  an  individual  who  has  recently  suffered  an  at- 
tack of  small-pox  or  scarlet  fever  contains  an  antitoxin 
which  would  neutralize  the  active  i^oison  of  the  disease 
in  the  circulation  of  another  i^erson  immediately  after 
infection.  Whether  a  small  quantity  of  blood  drawn 
from  the  veins  of  the  protected  individual  would  suffice 
to  arrest  the  progress  of  the  diseases  mentioned,  or  to 
modify  their  course,  can  only  be  decided  by  experiment ; 
but  the  experiment  seems  to  me  to  be  a  legitimate  one. 
Possibly  transfusion  of  a  moderate  amount  of  blood  from 
one  to  the  other  might  prove  to  be  curative,  or,  if  made 
in  advance  of  infection,  might  confer  immunity.  It  may 
be  that  an  antitoxin  can  be  obtained  from  the  blood  oi 
vaccinated  calves  which  would  have  a  curative  action  in 
small-pox." 

In  the  present  volume  the  author  has  endeavored  to 
give  a  summary  of  the  most  important  experimental  evi- 
dence in  the  field  of  research  to  which  it  relates.  Com- 
paratively little  space  has  been  given  to  the  discussion 
of  unsolved  questions  connected  with  the  subject ;  nor 
has  it  been  possible  to  review  the  entire  literature  ;  but 
the  most  important  results  of  experiments  made  by  com- 
petent bacteriologists  have  been  stated  as  concisely  as 
possible.  In  the  bibliography  the  titles  of  many  valuable 
papers  will  be  found  to  which  no  reference  has  been 
made  in  the  text. 

As  my  official  duties  occupy  the  greater  portion  of  my 
time  I  have  worked  under  a  certain  disadvantage,  but  it 
has  been  a  labor  of  love,  and  I  trust  that  the  result  will 
prove  to  be  acceptable  to  those  members  of  the  profes- 
sion who  wish  to  keep  abreast  of  the  progress  of  scien- 


VI 


tific  medicine  and  who  have  not  the  foreign  literature  at 
hand,  or  are  too  busy  to  make  use  of  it.  '"'-' 

In  many  places  throughout  the  work  I  have  introduced 
portions  of  the  text  of  my  "  Manual  of  Bacteriology,"  * 
and  of  some  of  my  recently  published  papers,  without  the 
use  of  quotation  marks. 


*  WUUam  Wood  &  Co.,  New  York,  1893. 


TABLE  OF  CONTENTS. 


PART   FIRST. 


IMMUNITY. 


I.  Natural  Immunity, 3 

II.  Acquired  Immunity, 34 

PART   SECOND. 

PROTECTIVE   INOCULATIONS   AND   SERUM-THEKAPY. 

I.  Anthrax, 75 

II.  Cluckeii  Cholera, 99 

III.  Cholera, Ill 

IV.  Diphtheria, 148 

V.  Foot-and-mouth  Disease, .172 

VI.  Glanders, 174 

VII.  Hog  Cholera,          .         . 182 

VIII.  Hog  Erysipelas, 186 

IX.  Hydrophobia, 192 

X.  Influenza 207 

XI.  Influenza  of  Horses, 209 

XII.  Pleuro-pneumonia  of  Cattle,         .....  211 

XIII.  Pneumonia, 216 

XIV.  Rinderpest, 227 

XV.  Small-pox, 228 

XVI.  Swine  Plague, 237 

XVII.  Streptococcus  Infection 241 

XVIII.  Symptomatic  Anthrax  ("Black  Leg"),         .         •         .245 

XIX.  Tetanus, 251 

XX.  Tuberculosis, 271 

XXI.  Typhoid  Fever, 292 

XXII.  Yellow  Fever, 301 

Index, 321 


PAET  FIEST. 
SUSCEPTIBILITY  AND  IMMUNITY. 


I. 

NATURAL  IMMUNITY. 

No  questions  in  general  biology  are  more  interesting, 
or  more  important,  from  a  practical  point  of  view,  than 
those  which  relate  to  the  susceptibility  of  certain  animals 
to  the  pathogenic  action  of  certain  species  of  bacteria, 
and  the  immunity,  natural  or  acquired,  from  such  patho- 
genic action  which  is  possessed  by  other  animals.  It  has 
long  been  known  that  certain  infectious  diseases,  now 
demonstrated  to  be  of  bacterial  origin,  prevail  only  or 
principally  among  animals  of  a  single  species.  Thus 
typhoid  fever,  cholera,  and  relapsing  fever  are  diseases  of 
man,  and  the  lower  animals  do  not  suffer  from  them  when 
they  are  prevailing  as  an  epidemic.  On  the  other  hand, 
man  has  an  immunity  from  many  of  the  infectious  diseases 
of  the  lower  animals,  and  diseases  of  this  class  which  pre- 
vail among  animals  are  frequently  limited  to  a  single 
species.  Again,  several  species,  including  man,  may  be 
susceptible  to  a  disease,  wliile  other  animals  have  a  nat- 
ural immunity  from  it.  Thus  tuberculosis  is  common  to 
man,  to  cattle,  to  apes,  and  to  small  herbivorous  animals 
by  inoculation,  while  the  carnivora  are,  as  a  rule,  im- 
mune ;  anthrax  may  be  communicated  by  inoculation  to 
man,  to  cattle,  to  sheep,  to  guinea-pigs,  rabbits,  and  mice, 
but  the  rat,  the  dog,  carnivorous  animals,  and  birds  are 
generally  immune ;  glanders,  which  is  essentially  a  dis- 
ease of  the  equine  genus,  may  be  communicated  to  man, 


4  IMMUNITY   AND   SERUM-TIIKKAPY. 

to  the  g-uiuea-pig",  and  to  field-mice,  while  house-mice, 
rabbits,  cattle,  and  swine  are  to  a  great  extent  im- 
mune. 

In  addition  to  this  general  race  immunity  or  suscepti- 
bility we  have  individual  differences  in  susceptibility  or 
resistance  to  the  action  of  pathogenic  bacteria,  which  may 
be  either  natural  or  acquired.  As  a  rule,  young  animals 
are  more  susceptihle  than  older  ones.  Thus  in  man  the 
young  are  especially  susceptible  to  scarlet  fever,  whoop- 
ing-cough and  other  "  children's  diseases,"  and  after  forty 
years  of  age  the  susceptibility  to  tubercular  infection  is 
very  much  diminished.  Among  the  lower  animals  it  is  a 
matter  of  common  laboratory  experience  that  the  very 
young  of  susceptible  species  may  be  infected  when  inocu- 
lated with  an  "  attenuated  culture  "  which  older  animals 
of  the  same  species  are  able  to  resist. 

Considerable  differences  as  to  susceptibility  may  also 
exist  among  adults  of  the  same  species.  In  man  these 
differences  in  individual  susceptibility  to  infectious  dis- 
eases are  frequently  manifested.  Of  a  number  of  persons 
exposed  to  infection  in  the  same  way  some  may  escape 
entirely,  while  others  have  attacks  differing  in  severity 
and  duration.  In  our  experiments  upon  the  lower  ani- 
mals we  constantly  meet  with  similar  results,  some  indi- 
viduals proving  to  be  exceptionally  resistant.  Excep- 
tional susceptibility  or  immur.ity  may  also  be  to  some 
extent  a  family  characteristic  or  one  of  race.  Thus  the 
negro  race  is  decidedly  less  subject  to  yellow  fever  than 
the  white  race,  and  this  disease  is  more  fatal  among  the 
fair-skinned  races  living  in  the  north  of  Europe  than 
among  the  Latin  races  living  in  trojiical  or  subtropical 
regions.  On  the  other  hand,  small -pox  appears  to  be  ex- 
ceptionally fatal  among  negroes. 


NATURAL   IMMUNITY.  5 

Avery  remarkable  instance  of  race  immunity  is  that  of 
Algerian  sheep  against  anthrax,  a  disease  which  is  very 
fatal  to  other  sheep. 

In  the  instances  mentioned  race  Immunity  is  probably 
an  acquired  tolerance  due  to  natural  selection  and  inher- 
itance. If,  for  examiDle,  a  susceptible  poi^ulation  is  ex- 
posed to  the  ravages  of  small-pox,  the  least  susceptible 
individuals  will  survive,  and  may  be  the  parents  of  chil- 
dren who  will  be  likely  to  inherit  the  special  characters 
as  regards  structure  or  physiological  activity  of  organs 
or  tissue  elements  upon  which  this  comparative  immu- 
nity depends.  The  tendency  of  continuous  or  repeated 
exposure  to  the  same  pathogenic  agent  will  evidently  be 
to  establish  a  race  tolerance  ;  and  there  is  reason  to  be- 
lieve that  such  has  been  the  effect  in  the  case  of  some  of 
the  infectious  diseases  of  man — e.g.,  syphilis,  small-pox — 
which  have  been  noticed  to  prevail  with  especial  severity 
when  first  introduced  among  a  virgin  population,  as  in 
the  islands  of  the  Pacific,  etc. 

In  the  same  way  we  may  explain  the  immunity  which 
carnivorous  animals  have  for  anthrax  and  various  forms 
of  septicaemia  to  which  the  herbivora  are  very  suscepti- 
ble when  the  pathogenic  germ  is  introduced  into  their 
bodies  by  inoculation.  From  time  immemorial  the  car- 
nivora  have  been  in  the  habit  of  fighting  over  the  dead 
bodies  of  herbivorous  animals,  some  of  Avliich  may  have 
fallen  a  prey  to  these  infectious,  germ  diseases,  and  in 
their  fighting  they  receive  wounds,  inoculated  Avith  the 
infectious  material  from  these  bodies,  which  would  be 
fatal  to  a  susceptible  animal.  If  at  any  time  in  the  past 
a  similar  susceptibility  existed  among  the  carnivora,  with 
individual  differences  as  to  resisting  power,  it  is  evident 
that  there  would  be  a  constant  tendency  for  the  most  sus- 


6  IMMUNITY   AND   SERUM-TIIEKAPY. 

ceptible  individuals  to  perish  and  for  the  least  suscepti- 
ble to  survive. 

The  essential  diflerence  between  a  susceptible  and  im- 
mune animal  depends  upon  the  fact  that  in  one  the  path- 
og-enic  germ,  when  introduced  by  accident  or  experi- 
mental inoculation,  multiplies  and  invades  the  tissues  or 
the  blood,  where,  by  reason  of  its  nutritive  requirements 
and  toxic  products,  it  produces  changes  in  the  tissues  and 
fluids  of  the  body  inconsistent  with  the  vital  requirements 
of  the  infected  animal ;  while  in  the  immune  animal  mul- 
tiplication does  not  occur,  or  is  restricted  to  a  local  inva- 
sion of  limited  extent,  and  in  which,  after  a  time,  the 
resources  of  nature  suffice  to  destroy  the  parasitic  in- 
vader. 

But  these  "resources  of  nature,"  iipon  which  natural 
immunity  depends,  are  not  at  all  times  available  for  the 
prevention  of  infection,  and  may  be  neutralized  by  vari- 
ous agencies  which  demand  consideration. 

It  has  been  shown  by  experiment  that  naturally  im- 
mune animals  may  be  infected  by  the  addition  of  certain 
substances  to  cultures  of  pathogenic  bacteria.  Thus 
Arloing  was  able  to  induce  symptomatic  anthrax  in  ani- 
mals naturally  immune  for  this  disease  by  mixing  with 
his  cultures  various  chemical  substances,  such  as  car- 
bolic acid,  pyrogallic  acid,  and  especially  lactic  acid 
(twenty  per  cent.).  Leo  has  shown  that  white  mice, 
which  are  not  subject  to  the  pathogenic  action  of  the 
glanders  bacillus,  may  be  rendered  susceptible  by  feed- 
ing them  for  some  time  upon  phloridzin,  which  gives 
rise  to  an  artificial  diabetes,  and  causes  the  tissues  to 
become  impregnated  with  sugar.  Behring  claims  to 
have  demonstrated  by  experiment  that  white  rats  lose 
their  immunity  for  anthrax  when  fed  for  some  time  upon 


ISTATURAL   IMMUNITY.  7 

an  exclusively  vegetable  diet,  or  when  phosphate  of  lime 
is  added  to  their  food,  and  he  has  suggested  that  the  im- 
munity of  these  animals  may  be  due  to  the  highly  alka- 
line reaction  of  their  blood  and  tissue  juices. 

Bouchard  has  shown  that  very  small  doses  of  a  pure 
culture  of  Bacillus  pyocyaneus  are  fatal  to  rabbits  when 
at  the  same  time  a  considerable  quantity  of  a  filtered 
culture  of  the  same  bacillus  is  injected  into  a  vein.  The 
animal  could  have  withstood  the  filtered  culture  alone,  or 
the  bacillus  injected  beneath  its  skin  ;  but  its  resisting 
power — natural  immunity — is  overcome  by  the  combined 
action  of  the  living  bacilli  and  the  toxic  substances  con- 
tained in  the  filtered  culture.  The  same  result  may  be 
obtained  by  injecting  sterilized  cultures  of  a  different 
microorganism.  Thus  Roger  has  shown  that  the  rabbit, 
Avhich  has  a  natural  immunity  against  symptomatic  an- 
thrax, succumbs  to  infection  when  inoculated  with  a  cult- 
ure of  the  bacillus  of  this  disease,  if  at  the  same  time  it 
receives  an  injection  of  a  sterilized  or  non-sterilized  cult- 
ure of  Bacillus  prodigiosus.  Monti  has  succeeded  in 
killing  animals  with  old  and  attenuated  cultures  of 
Streptococcus  pyogenes,  or  of  Staphylococcus  pyogenes 
aureus,  by  injecting  at  the  same  time  a  culture  of  Pro- 
teus vulgaris.  In  a  similar  way,  it  seems  probable,  the 
normal  resistance  of  man  to  infection  by  certain  patho- 
genic bacteria  may  be  overcome.  Thus  when  water  con- 
taminated by  the  i^resence  of  the  typhoid  bacillus  is 
used  for  drinking  by  the  residents  of  a  certain  town  or 
district,  not  all  of  those  who  in  this  way  are  exposed  to 
infection  contract  typhoid  fever,  and  among  those  who 
do,  there  is  good  reason  to  believe  that,  in  certain  cases 
at  least,  the  result  depends  upon  an  additional  factor  of 
the  kind  suggested  by  the  above-mentioned  experiments, 


8  IMMUNITY   AND   SERUM-THERAPY. 

e.g.,  the  consumption  of  food  containing  putrefactive 
products  iDi'oduced  by  Proteus  vulg-aris  and  other  sapro- 
phytes, or  the  respiration  of  an  atmosphere  containing 
the  volatile  products  of  putrefaction  {e.g.,  sewer-gas). 
Recent  experiments  (1894)  made  by  Alessi,  in  the  Hy- 
gienic Institute  of  the  University  of  Eome,  give  support 
to  this  view.  The  experiments  were  made  upon  rats, 
guinea-pigs,  and  rabbits.  The  rats  were  confined  in  a 
close  cage  with  perforated  bottom,  which  was  placed 
over  the  opening  of  a  privy  ;  the  guinea-pigs  and  rabbits 
in  similar  cages  having  a  receptacle  below  in  which  their 
own  excreta  was  allowed  to  accumulate.  The  animals 
which  breathed  an  atmosphere  vitiated  in  this  way  lost, 
after  a  time,  their  usual  activity  and  became  emaciated, 
although  the}'  continued  to  eat  greedily.  When  these 
animals  were  inoculated  with  a  small  quantity  of  a  cult- 
ure of  the  typhoid  bacillus  (0.25  to  0.5  c.c),  they  died 
wdthiu  from  twelve  to  thirty-six  hours.  The  same 
amount  of  the  typhoid  culture  injected  into  control  ani- 
mals produced  no  injurious  effect.  In  the  animals  which 
succumbed  to  typhoid  infection  there  was  found  a  hemor- 
rhagic enteritis,  increase  in  volume  of  Peyer's  glands  and 
of  the  spleen,  and  typhoid  bacilli  in  the  blood,  liver,  and 
spleen.  The  characteristic  appearances  of  typhoid  in- 
fection were  more  pronounced  in  the  rabbits  and  guinea- 
pigs  than  in  rats.  Similar  experiments  with  Bacillus 
coli  communis  gave  similar  results.  The  time  required 
to  induce  this  predisposition  for  typhoid  infection  was 
from  five  to  seventy-two  daj's  for  the  rats,  seven  to  fifty- 
eight  for  the  guinea-pigs,  and  three  to  eighteen  for  the 
rabbits.  Alessi  found  that  the  susceptibility  to  infection 
diminished  after  a  certain  time,  and  suggests  that  in  a 
similar  wa}'  man  may  become  habituated  to  breathing  an 


NATURAL   IMMUNITY.  9 

atmosphere  containing"  sewer-gases,  which  for  those  un- 
accustomed to  it  wouki  be  likely  to  induce  typhoid  infec- 
tion in  case  the  typhoid  bacillus  should  be  introdiiced 
into  the  intestine.  An  attempt  to  determine  what  par- 
ticular gas  was  concerned  in  the  neutralization  of  the 
natural  immunity  of  the  animals  experimented  upon  was 
not  successful.  The  following-  g-ases,  tested  separately, 
gave  a  negative  result :  Ammonia,  hydrogen  sulphide,  car- 
bon dioxide,  carbonic  oxide,  and  ammonium  sulphide. 
The  natural  immunity  of  healthy  animals  may  also  be 
neutralized  by  other  agencies  which  have  a  depressing 
effect  upon  the  vital  resisting  power.  Thus  Nocard  and 
Eoux  found  by  experiment  that  an  attenuated  cvilture  of 
the  anthrax  bacillus,  which  was  not  fatal  to  guinea-pigs, 
killed  these  animals  when  injected  into  the  muscles  of 
the  thigh  after  they  had  been  bruised  by  mechanical  vio- 
lence. Abarrin  and  Roger  found  that  white  rats,  which 
are  not  susceptible  to  anthrax,  became  infected  and  fre- 
quently died  if  they  were  exhausted,  previous  to  inocula- 
tion, by  being  compelled  to  turn  a  revolving  wheel  for  a 
considerable  time.  Pasteur  found  that  fowls,  which 
have  a  natural  immunity  against  anthrax,  become  in- 
fected and  perish  if  they  are  subjected  to  artificial  re- 
frigeration after  inoculation.  This  has  been  conifirmed 
by  the  more  recent  experiments  of  Wagner  (1891).  Ac- 
cording to  Canalis  and  Morpurgo,  pigeons  which  are  en- 
feebled by  inanition  easily  contract  anthrax  as  a  result 
of  inoculation.  Arloing  states  that  sheep  which  have 
been  freely  bled  contract  anthrax  more  easily  than 
others ;  and  Serafini  found  that  when  dogs  were  freely 
bled  the  bacillus  of  Friedlander,  injected  into  the  trachea 
or  the  pleural  cavity,  entered,  and  apparently  multiplied 
to  some  extent  in  the  blood,  whereas  without  such  pre- 


10  IMMUNITY   AND   SERUM-THEKAPY. 

vioiis  bleeding"  they  were  not  to  be  found  in  the  circu- 
lating fluid.  Certain  anaesthetic  agents  have  also  been 
shown  to  produce  a  similar  result.  Platania  communi- 
cated anthrax  to  immune  animals — dogs,  frogs,  pigeons 
— by  bringing  them  under  the  influence  of  curare,  chloral, 
or  alcohol ;  and  Wagner  obtained  similar  results  in  his 
experiments  upon  pigeons  to  which  he  had  administered 
chloral.  In  man,  clinical  experience  shows  that  those 
Avho  are  addicted  to  the  excessive  use  of  alcohol  are  es- 
pecially liable  to  contract  certain  infectious  diseases — 
pneumonia,  erysipelas,  yellow  fever,  etc. 

The  micrococcus  of  pneumonia  is  habitually  present  in 
the  salivary  secretions  of  many  healthy  individuals,  and 
it  is  evident  that  an  attack  of  pneumonia  does  not  de- 
])end  alone  upon  the  presence  of  this  micrococcus,  which 
has,  nevertheless,  been  conclusivelj^  shown  to  be  the 
usual  infectious  agent  in  cases  of  croupous  pneumonia. 
No  "doubt  the  introduction  of  the  pathogenic  micrococcus 
to  the  vulnerable  point — the  lungs — is  an  essential  factor 
in  the  development  of  a  case  of  pneumonia,  but  there 
is  reason  to  believe  that  there  are  other  factors  equally 
essential.  Thus  it  is  well  known  that  an  attack  of  pneu- 
monia often  results  from  exposure  to  cold,  which  may 
act  as  an  exciting  cause  ;  and,  also,  that  a  recent  attack 
of  an  acute  febrile  disease — especially  measles — consti- 
tutes a  predisposing  cause.  It  is  generallj^  recognized 
that  malnutrition,  want  of  exercise,  insanitary  surround- 
ings, and  continued  respiration  of  an  atmosphere  loaded 
with  dust,  as  in  cotton-mills,  or  a  recent  attack  of  pneu- 
monia, constitute  predisposing  causes  to  tubercular  in- 
fection by  way  of  the  lungs. 

AVliile  natural  immunity  may  be  overcome  by  the  vari- 
ous depressing  agencies  referred  to,  it  is  also  true  that  it 


NATURAL   IMMUNITY.  11 

lias  only  a  relative  value  in  the  absence  of  these  predis- 
posing causes,  and  may  be  overcome  by  unusual  viru- 
lence of  the  pathogenic  infectious  agent,  or  by  the  intro- 
duction into  the  body  of  an  excessive  amount  of  a  pure 
culture  of  the  same. 

The  pathogenic  potency  of  known  disease  germs  varies 
as  widely  as  does  the  susceptibility  of  individuals  to 
their  specific  action.  In  general  it  may  be  said  that  the 
more  recently  the  germ  comes  from  a  developed  case  of 
the  disease  to  which  it  gives  rise,  the  more  virulent  it  is, 
and  the  longer  it  has  been  cultivated  outside  of  the  ani- 
mal body  the  more  attenuated  is  its  pathogenic  power. 
Thus  when  the  discharges  of  a  typhoid-fever  patient  find 
their  way  directly  to  a  Avater-supply  of  limited  amount  a 
large  proportion  of  those  who  drink  the  water  are  likely 
to  be  attacked ;  but  when  a  considerable  interval  of  time 
has  elapsed  since  the  contamination  occurred,  although 
the  germs  may  still  be  present,  the  liability  to  attack  is 
much  less  on  account  of  diminished  pathogenic  viru- 
lence. 

The  development  of  an  attack  also  depends,  to  some 
extent,  upon  the  number  of  germs  introduced  into  a  sus- 
ceptible individual  at  one  time.  The  resources  of  nature 
may  be  sufficient  to  dispose  of  a  few  bacilli,  while  a  large 
number  may  overwhelm  the  resisting  power  of  the  indi- 
vidual. 

The  experiments  of  Cheyne  (1886)  show  that  in  the 
ease  of  very  pathogenic  species,  a  single  bacillus,  or  at 
least  a  very  small  number,  introduced  beneath  the  skin, 
may  produce  fatal  infection  in  a  very  susceptible  animal, 
while  greater  numbers  are  required  in  those  less  suscep- 
tible. Thus  a  guinea-pig  succumbed  to  general  infec- 
tion after  being  inoculated  subcutaneouslv  with  antlirax 


12  IMMUNITY   AND   SERUM-TIIEnAPY. 

blood  diluted  to  such  an  extent  that,  by  estimation,  only 
one  bacillus  was  present  in  the  fluid  injected ;  and  a 
similar  result  was  obtained  in  mice  with  Bacillus  muri- 
septicus.  In  the  case  of  the  microbe  of  fowl  cholera 
(Bacillus  septicaemia  hemorrhagicse),  Cheyne  found  that 
for  rabbits  the  fatal  dose  was  300,000  or  more,  that  from 
10,000  to  300,000  cause  a  local  abscess,  and  that  less  than 
10,000  produce  no  appreciable  effect.  The  common  sa- 
prophyte, Proteus  vulgaris,  was  found  to  be  pathogenic 
for  rabbits  when  injected  into  the  dorsal  muscles  in  suf- 
ficient numbers.  But,  according  to  the  estimates  made, 
225,000,000  were  required  to  cause  death,  while  doses  of 
from  9,000,000  to  112,000,000  produced  a  local  abscess, 
and  less  than  9,000,000  gave  an  entirely  negative  result. 

In  the  scale  of  living  things  man  stands  at  the  head, 
and  the  unicellular  organisms  known  as  bacteria  at  the 
foot,  yet  the  relations  of  these  "microbes"  to  the  "lord 
of  creation  "  are  more  important  and  more  complex  than 
those  which  exist  between  man  and  any  other  single 
group  of  living  organisms. 

In  the  absence  of  bacteria,  or  of  some  other  organisms 
to  performs  their  functions,  the  continued  existence  of 
man  upon  the  face  of  the  globe  would  be  impossible  ; 
for  it  is  due  to  their  activity  in  the  decomposition  of 
animal  and  vegetable  substances  that  organic  material, 
stored  up  as  a  result  of  the  vital  activities  of  higher  plants 
and  animals,  is  returned  to  the  soil  and  atmosphere  after 
the  death  of  these.  Without  such  a  provision  of  nature 
organic  life  would  long  since  have  come  to  a  standstill, 
from  the  storing  up  of  those  essential  elements  which  go 
to  make  up  the  structure  of  animal  and  vegetable  tissues. 
But  as  a  result  of  the  decomposition  of  such  tissues  after 
death,  through  the  agency  of  bacteria,  these  elements 


NATURAL   IMMUNITY.  13 

are  released  from  tlie  complex  combinations  in  which 
they  exist  and  again  become  available  for  the  nutrition 
of  living  plants  and  animals.  The  moment  life  is  extinct, 
when  temperature  conditions  are  favorable,  these  de- 
structive processes  commence.  The  saprophytic  bacte- 
ria, constantly  present  in  the  alimentary  canal,  quickly 
invade  the  tissues,  and  soon  the  whole  body  is  invaded 
by  these  agents  of  putrefactive  decomposition.  But  the 
living  body  not  only  resists  the  invasion  of  these  organ- 
isms through  the  walls  of  the  intestine  and  the  external 
integument,  but  is  able  to  dispose  of  a  considerable  num- 
ber of  putrefactive  microorganisms  when  these  are  in- 
jected into  the  circulation.  Now,  to  some  extent  at  least, 
the  resistance  to  invasion  by  pathogenic  bacteria — natu- 
ral immunity — corresponds  with  the  resistance  to  the 
putrefactive  organisms  referred  to,  and  no  doubt  depends 
upon  similar  causes.  Some  of  these  pathogenic  bacteria 
are  very  commonly  found  upon  the  surface  of  the  body 
of  healthj^  individuals  or  upon  exposed  mucous  mem- 
branes. As  already  mentioned,  the  micrococcus  of  croup- 
ous pneumonia  is  very  frequently  found  in  the  salivary 
secretions  of  healthy  persons,  and  it  seems  probable  that 
its  normal  habitat  is  the  human  mouth.*  The  pus  cocci 
— Staphylococcus  aureus  and  albus — are  not  infrequently 
found  in  the  mouths  or  on  the  surface  of  the  body  of 
healthy  persons.f    And  during  the  prevalence  of  diphthe- 


*  Netter  found  it  in  the  salivary  secretions  of  fifteen  per  cent,  of  the  healthy 
individuals  examined — one  hundred  and  sixty-five  in  all. 

t  Netter  found  Staphylococcus  aureus  seven  times  in  the  salivary  secretions, 
out  of  one  hundred  and  twenty-seven  individuals  examined.  Steffeck  (1892) 
examined  the  vaginal  secretions  of  twenty-nine  pregnant  females,  and  found 
S.  pyogenes  albus  in  nine,  S.  pyogenes  aureus  in  three,  and  Streptococcus  pyo- 
genes in  one.  In  nasal  mucus  Von  Besser,  in  eighty-one  cases  examined,  found 
"diplococcus  pneumoniae"  fourteen  times,  S.  pyogenes  aureus  fourteen  times, 
and  Streptococcus  pyogenes  seven  times. 


14  IMMUNITY    AND    SERUM-THERAPY. 

ria  in  a  household,  diphtheria  bacilli  have  been  found  in 
the  mouths  of  individuals  who  presented  no  evidence  of 
diphtheria  infection.  When  the  normal  resisting-  power 
of  the  tissues  is  overcome  locally  by  mechanical  vio- 
lence, by  arrest  of  circulation,  by  freezing,  etc.,  the  ordi-. 
nary  putrefactive  bacteria  may  invade  the  injured  tis- 
sues, and  as  a  result  we  have  gangrene.  Or  an  invasion 
by  pus  cocci  may  give  rise  to  a  localized  inflammation, 
resulting  in  abscess  formation.  Again,  certain  condi- 
tions relating  to  the  diet  and  surroundings  of  an  indi- 
vidual may  constitute  a  predisposition  to  local  infectious 
processes  of  greater  or  less  extent,  due  to  invasion  of  the 
tissues  by  the  common  pathogenic  micrococci  above  re- 
ferred to.  Thus  we  have  developed  acne  pustules,  boils, 
carbuncles,  etc. 

Explanation  of  Natural  Immunity. 

We  have  now  to  inquire  upon  what  the  natural  immu- 
nity depends  which  enables  the  healthy  animal  body  to 
resist  invasion  by  the  destructive  agents  referred  to. 

Phagocytosis. — In  my  chapter  on  "  Bacteria  in  Infec- 
tious Diseases,"  in  "  Bacteria,"  published  in  the  spring 
of  1884,  but  placed  in  the  hands  of  the  publishers  in 
1883,  I  say : 

"  It  may  be  that  the  true  explanation  of  the  immunity 
afforded  by  a  mild  attack  of  an  infectious  germ  disease 
is  to  be  found  in  an  acquired  tolerance  to  the  action  of  a 
chemical  poison  produced  by  the  micro-organism,  and 
consequent  ability  to  bring  the  resources  of  nature  to 
bear  to  restrict  invasion  by  the  parasite." 

In  the  same  chapter  the  resources  of  nature  supposed 
to  be  brought  to  bear  in  restricting  invasion  by  the  para- 
site are  referred  to  as  follows  : 


NATURAL   IMMUNITY.  15 

"  If  we  add  a  small  quantity  of  culture-fluid  containing- 
the  bacteria  of  putrefaction  to  the  blood  of  an  animal 
withdrawn  from  the  circulation- into  a  proper  receptacle 
and  maintained  in  a  culture-oven  at  blood-heat,  we  will 
find  that  these  bacteria  multiply  abundantly,  and  evi- 
dence of  13 utref active  decomposition  will  soon  be  per- 
ceived. But  if  we  inject  a  like  quantity  of  the  culture- 
fluid,  with  its  contained  bacteria,  into  the  circulation  of 
a  living-  animal,  not  only  does  no  increase  and  no  putre- 
factive change  occur,  but  the  bacteria  introduced  quickly 
disappear,  and  at  the  end  of  an  hour  or  two  the  most 
careful  microscopical  examination  will  not  reveal  the 
presence  of  a  single  bacterium.  This  difference  we  as- 
cribe to  the  vital  properties  of  the  fluid  as  contained  in 
the  vessels  of  a  living  animal,  and  it  seems  probable  that 
the  little  masses  of  protoplasm  known  as  white  blood- 
corpuscles  are  the  essential  histological  elements  of  the 
blood,  as  far  as  any  manifestation  of  vitality  is  con- 
cerned. The  writer  has  elsewhere  (1881)  svygested  that  the 
disappearance  of  the  hactevia  frotn  the  circulation,  in  the  ex- 
periment referred  to,  may  he  effected  hj  the  white  corpuscles, 
which,  it  is  well  known,  pick  up,  after  the  manner  of 
amoebee,  any  particles,  org-anic  or  inorg-anic,  which 
come  in  their  way.  And  it  requires  no  great  stretch  of 
credulity  to  believe  that  theif  may,  like  an  amoeba,  digest 
and  assimilate  the  p>votoplasin  of  the  cajjfnred  bacterium, 
thus  putthig  an  end  to  the  possibility  of  its  doing  any  harm. 

"  In  the  case  of  a  pathogenic  organism  we  may  imag- 
ine that,  when  captured  in  this  way,  it  may  share  a  like 
fate  if  the  captor  is  not  paralyzed  by  some  potent  poison 
evolved  by  it,  or  overwhelmed  by  its  superior  vigor  and 
rapid  multiplication.  In  the  latter  event  the  active  ca- 
reer of  our  conservative  white  corpuscles  would  be  quickly 
terminated  and  its  protoplasm  would  serve  as  food  for 
the  enemy.  It  is  evident  that  in  a  contest  of  this  kind 
the  balance  of  power  would  depend  upon  circumstances 
relating  to  the  inherited  vital  characteristics  of  the  invad- 
ing parasite  and  of  the  invaded  leucocyte." 


16  IMMUNITY   AND   SERUM-THERAPY. 

This  explanation  is  now  very  commonly  spoken  of  as 
the  "  Metschuikoff  tlieoiy,"  althoiig-h,  as  a  matter  of  fact, 
it  was  clearly  stated  by  the  writer  several  years  (1881) 
before  Metschuikofl"s  first  j^aper  (1884)  was  published. 
Metschnikoff  has,  however,  been  the  principal  defender 
of  this  explanation  of  acquired  immunity,  and  has  made 
extensive  and  painstaking  researches,  as  a  result  of 
which  many  facts  have  been  brought  to  light  which  ajD- 
l^ear  to  give  support  to  the  present  writer's  hypothesis — 
the  so-called  Metschnikoff  theor\\ 

The  observations  which  first  led  Metschuikoff  to  adopt 
the  explanation  of  immunity  under  consideration  were 
made  u^Don  a  species  of  daphuia,  which  is  subject  to  in- 
fection by  a  torula  resembling  the  yeast  fungus.  Enter- 
ing with  the  food,  this  fungus  penetrates  the  walls  of  the 
intestine  and  invades  the  tissues.  In  certain  cases  the 
infection  does  not  prove  fatal,  owing,  as  Metschnikoff  as- 
serts, to  the  fact  that  the  fungus  cells  are  seized  upon  by 
the  leucocytes,  which  appear  to  accumulate  around  the 
invading  parasite  (chemiotaxis)  for  this  special  purpose. 
If  they  are  successful  in  overpowering  and  destroying 
the  parasite,  the  animal  recovers  ;  if  not,  it  succumbs  to 
the  general  infection  which  results.  In  a  similar  man- 
ner, Metschnikoff'  supposes,  pathogenic  bacteria  are  de- 
stroyed when  introduced  into  the  body  of  an  immune 
animal.  The  colorless  blood-corpuscles  which  he  de- 
nominates j)hagocytes,  accumulate  at  the  point  of  inva- 
sion and  pick  up  the  bacteria,  as  they  are  kno^Ti  to  pick 
up  inorganic  particles  injected  into  the  circulation.  So 
far  there  can  be  no  doubt  that  Metschnikoff'  is  right. 
The  presence  of  bacteria  in  the  leucocytes  in  considera- 
ble numbers,  both  at  the  point  of  inoculation  and  in  the 
general  circulation,   has  been  repeatedly   demonstrated 


NATUEAL   IMMUNITY.  17 

in  animals  inoculated  with  various  pathogenic  bacteria. 
The  writer  observed  this  in  his  experiments,  made  in 
1881,  in  which  rabbits  were  inoculated  with   cultures  of 
his  "  Micrococcus  Pasteuri  "    (micrococcus  of   croupous 
pneumonia),  and  it  was  this  observation  which  led  him 
to  suggest  the  hypothesis  which  has  since  been  so  vig- 
orously supported  by  Metschnikoff.     But  the  presence  of 
a  certain  number  of  bacteria  within  the  leucocytes  does 
not  prove  the  destructive  power  of  these  cells  for  living 
pathogenic  organisms.     As  urged  by  Weigert,  Baumgar- 
ten,  and  others,  it  may  be  that  the  bacteria  were  already 
dead  when  they  were  picked  up,  having  been  destroyed 
by  some  agency  outside  the  blood-cells  ;  and,  as  we  shall 
see   later,   there   is    experimental   evidence   that   blood- 
serum  has  decided  germicidal  power  for  certain  patho- 
genic bacteria,  and  that  the  blood-serum  of  the  rat  and 
other   animals,  which   have  a  natural  immunity   against 
anthrax,  is  especially  fatal  to  the  anthrax  bacillus.     On 
the  other  hand,  there  is  reason  to  believe  that  living  bac- 
teria picked  up  by  leucocytes  are  not  always  destroyed, 
but  that,  in  some  instances  at  least,  they  thrive  and  mul- 
tiply within  these  protoplasmic  masses.     In  mouse  sep- 
ticaemia  and   in   gonorrhoea    one  would  be  disposed  to 
decide,  from  the  appearance  and  arrangement  of  the  pa- 
thogenic bacteria  in  the  leucocytes,  that  tliey  are  not  de- 
stroyed but  that  they  multiply  in  the  interior  of  these 
cells,  which  in  the  end  succumb  to  this  parasitic  inva- 
sion.    In  both  of  the  diseases  mentioned  we  find  the  leu- 
cocytes so  completely  filled  with  the  pathogenic  micro- 
organisms that  it  is  difficult  to  believe  that  they  have 
all  been  picked  up  by  a  voracious  phagocyte,  which  has 
stuffed  itself  to  repletion,  while  numerous   other  leuco- 
cj'^tes  in  the  same  microscopic  field  have  failed  to  capture 


18  IMMUNITY    AND   SERUM-THEIIA  1>Y. 

a  single  bacillus  or  micrococcus.  Moreover,  the  staining 
of  the  parasitic  invaders,  and  the  characteristic  arrange- 
ment of  the  "  gonococcus  "  in  stained  preparations  of 
gonorrhoeal  pus,  indicate  that  their  vitality  has  not  been 
destroyed  in  the  interior  of  the  leucocytes  or  pus-cells, 
and  we  can  scarcely  doubt  that  the  large  number  found 
in  cei-tain  cells  is  due  to  multiplication  ///  sita  rather  than 
to  an  unusual  activity  of  these  particular  cells.  But  in 
certain  infectious  diseases,  and  especially  in  inoculations 
of  the  anthrax  bacillus  into  immune  animals,  the  bacilli 
included  within  the  leucocytes  often  give  evidence  of  de- 
generative changes,  which  would  support  the  view  that 
they  are  destroyed  within  the  leucocytes,  unless  these 
changes  occurred  before  they  were  picked  up,  as  claimed 
by  Nuttall  and  others. 

Metschnikoif  divides  the  so-called  phagocytes  into  two 
grouj)s :  Fixed  phagocytes  (endothelial  cells,  etc.)  and 
free  phagocytes.  According  to  his  observations  not  all 
leucocytes  are  phagocytes.  The  comparatively  small, 
immobile  leucocytes  ("  lymphocytes ")  with  a  single 
large  nucleus  never  take  up  bacteria.  The  large  uninu- 
clear leucocyte  which  exhibits  active  amoeboid  move- 
ments, and  in  which  the  nucleus  is  frequently  lobed  or 
reniform,  is  called  by  Metschnikoff  a  macrophage,  and  the 
smaller  multinuclear  leucocyte  (or  with  one  nucleus  in 
process  of  breaking  up)  he  calls  a  microphage.  The 
macrophages  are  believed  to  be  largely  of  endothelial 
origin.  Metschnikoff  asserts  that  in  general  the  more 
virulent  the  microorganism  the  rarer  is  its  presence  ob- 
served within  the  iDhagocytes.  Thus  in  the  acute  sep- 
ticaemias which  are  quickly  fatal  to  siisceptible  animals — 
e.g.,  fowl  cholera,  rabbit  septicaemia,  anthrax  in  mice,  etc. 
— the  pathogenic  bacteria  are  rarely  found  in  the   inte- 


NATURAL   IMMriNITY,  19 

rior  of  the  cells.  They  remain  free  in  the  vicinity  of  the 
point  of  inoculation  and  thence  quickly  invade  the 
blood.  On  the  other  hand,  in  infectious  diseases  having- 
a  more  protracted  course  there  is  a  very  decided  phago- 
cytosis. As  an  example  of  this  Metschnikoff  mentions 
mouse  septicaemia  "  which  has  a  duration  in  the  mouse 
two  and  a  half  times  as  long-  as  that  of  anthrax  in  the 
same  animal,"  and  the  chronic  infectious  diseases — tuber- 
culosis, leprosy,  rhinoscleroma,  and  g-landers.  Ag-ain,  at- 
tention is  called  to  the  phenomena  attending  recovery 
from  certain  infectious  diseases.  In  relapsing-  fever,  for 
example,  during  the  sudden  access  of  the  fever  the  spi- 
rilla are  present  in  the  blood  in  great  numbers,  and  they 
are  not  included  within  the  cells.  During  the  apyretic 
stage  they  disappear  from  the  blood,  but  are  found  in- 
cluded in  the  phagocytes  of  the  spleen.  In  like  manner 
rats  and  pigeons  which  survive  an  attack  of  anthrax, 
when  killed  during  the  period  of  resolution  show  numer- 
ous bacilli  included  within  leucocytes  and  splenic  phago- 
cytes, and  very  few  free  bacilli. 

In  a  similar  manner,  according  to  Metschnikoff,  ani- 
mals which  have  an  acquired  immunity  for  anthrax  or 
other  infectious  diseases  of  bacterial  origin,  when  inocu- 
lated subcutaneously,  resist  infection  because  there  is  an 
extensive  emigration  of  leucocytes  to  the  jDoint  of  inocu- 
lation, and  these  soon  include  the  bacteria  in  large  num- 
bers. In  susceptible  animals,  not  having  thus  acquired 
immunity,  the  phagocytes  are  inoperative,  and  the  bac- 
teria are  very  rarely  seen  included  within  them.  As  ex- 
amples of  this  Metschnikoff  cites  the  bacillus  of  anthrax 
and  Vibrio  Metschnikovi.  In  unvaccinated  rabbits  in- 
oculated with  anthrax,  phagocytosis  is  said  to  be  very 
seldom  seen,  while  in  rabbits  protected  by  "  vaccination  " 


20  IMMUNITY   AND   SERUM-THKKAPY. 

it  is  veiy  marked.  The  differance  is  still  more  remarka- 
ble in  guiDea-pigs  inoculated  with  Vibrio  Metschnikovi. 
In  vaccinated  animals  the  phagocytes  are  loaded  with 
the  microbes,  in  those  not  vaccinated  they  are  not  seen 
included  in  these  cells.  According  to  Metschnikoff  the 
giant  cells  of  tuberculosis  are  "  huge  multinuclear  phago- 
cj'tes  "  in  which  the  bacilli  are  destroyed.  This  is  shown 
by  the  "  very  evident  signs  of  degeneration  ;  the  bacilli 
swell,  their  enveloping  membrane  becomes  much  thick- 
ened and  highly  refractive,  and  in  time  the  contents  lose 
the  power  of  fixing  the  staining  material,  so  that,  event- 
ually, nothing  is  left  but  slightl}^  yellowish  forms,  re- 
calling, in  propoiiions  and  position,  the  enlarged  ba- 
cilli." 

Actio)i  of  Blood-Serum  and  OtJier  Organic  Liquids  Upon 
Bacteria. — Bacteriologists  have  long  been  aware  of  the 
fact  that  many  species  of  bacteria,  when  injected  into  the 
circulation  of  a  living  animal,  soon  disappear  from  the 
blood,  and  that  the  blood  of  such  an  animal  a  few  hours 
after  the  intravenous  injection,  of  putrefactive  bacteria 
for  example,  does  not  contain  living  bacteria  capable  of 
development  in  a  suitable  culture  medium.  Wyssoko- 
witsch,  in  an  extended  series  of  experiments,  has  shown 
that  non -pathogenic  bacteria  may  be  obtained  in  cultures 
from  the  liver,  spleen,  kidneys,  and  bone  marrow  after 
they  have  disappeared  from  the  blood  ;  but  that,  as  a 
rule,  those  x^resent  in  these  organs  have  lost  their  vital- 
it}^  as  shown  by  culture  experiments,  in  a  period  varying 
from  a  few  hours  to  two  or  three  days.  As  has  been 
stated  above,  this  disappearance  was  accounted  for  by 
the  writer  (1881),  and  later  by  Metschnikoff  (1884),  as  the 
result  of  the  vital  activities  of  the  leucocytes  ;  but  more 
recently  experimental  evidence  has  been  presented  which 


NATURAL   IMMUNITY.  21 

indicates  that  tliis  is  not  the  only,  and  probably  not  the 
principal,  agency  by  which  bacteria  introduced  into  the 
circulation  of  living  animals  are  destroyed. 

Von  Fodor  (1887)  first  called  attention  to  the  fact  that 
anthrax  bacilli  may  be  destroyed  by  freshly  drawn  blood  ; 
and  Nuttall  (1888),  in  an  extended  series  of  experiments, 
showed  that  various  bacteria  are  destroyed  within  a 
short  time  by  the  fresh  blood  of  warm-blooded  animals. 
Thus  the  anthrax  bacillus  in  rabbit's  blood  was  usually 
killed  in  from  two  to  four  hours  when  the  temperature 
Avas  maintained  at  37°  to  38°  C,  and  the  same  result  was 
obtained  with  pigeon's  blood  at  41°  C.  But  when  the 
blood  was  allowed  to  stand  for  some  time,  or  was  sub- 
jected to  a  temperature  of  55°  C,  it  no  longer  joossessed 
germicidal  properties  and  served  as  a  culture-fluid  in 
which  an  abundant  development  of  anthrax  bacilli  oc- 
curred. Bacillus  subtilis  and  Bacillus  megatherium  were 
also  destroyed  by  fresh  rabbit's  blood,  but  it  was  without 
action  upon  Staphylococcus  pj^ogenes  aureus,  which,  at  a 
temperature  of  37.5°  C,  was  found  to  have  increased  in 
numbers  at  the  end  of  two  hours.  Further  researches  by 
Behring  and  Nissen  (1890)  show  that  there  is  a  wide  dif- 
ference in  the  blood  of  different  animals  as  to  germicidal 
power,  and  that  certain  bacteria  are  promptly  destroyed, 
while  other  species  are  simply  restrained  for  a  time  in 
their  development  or  are  not  affected.  Thus  Nissen 
found  that  the  cholera  spirillum,  the  bacillus  of  anthrax, 
the  bacillus  of  typhoid  fever,  and  Friedliinder's  bacillus 
were  killed,  while  Staphylococcus  pyogenes  albus  and 
aureus.  Streptococcus  pyogenes,  the  bacillus  of  foAvl 
cholera  (Bacillus  septicaemiae  haemorrhagicge),  the  bacil- 
lus of  rothlauf  and  Proteus  hominis  were  able  to  mul- 
tiply in  rabbit's  blood  after  having  been  restrained  for 


22  IMMUNITY    AND   SERUM-TIIEUAPY. 

a  short  time  in  their  development.  In  the  case  of  the 
cholera  spirillum  a  period  of  ten  to  forty  minutes  suf- 
ficed for  the  complete  destruction  of  a  limited  number, 
but  when  the  number  exceeded  1,200,000  per  c.c.  they 
were  no  longer  destroyed  with  certaint}^  and  after  five 
hours  an  increase  occurred.  The  anthrax  bacillus  was 
commonly  destroyed  within  twentj^  minutes,  and  the 
typhoid  bacillus  at  the  end  of  two  hours.  In  the  experi- 
ments of  Behring-  and  Nissen  it  was  found  that  the  most 
pronounced  germicidal  eflect  was  obtained  from  the 
blood  of  the  rat,  an  animal  which  has  a  natural  immunity 
ag-ainst  anthrax ;  while  the  blood  of  the  g-uinea-pig-,  a 
verj^  susceptible  animal,  had  no  restraining  effect  and 
served  as  a  favorable  culture-medium  for  the  anthrax  ba- 
cillus. And  the  remarkable  fact  was  developed  that  when 
the  blood  of  the  rat  was  added  to  the  blood  of  the  guinea- 
XJig",  in  the  proportion  of  1  to  8,  it  exercised  a  decided 
restraining-  influence  on  the  growth  of  the  anthrax  bacil- 
lus. Later  researches  have  shown  that  cultivation  in  the 
blood  of  an  immune  animal  causes  an  attenuation  of  the 
virulence  of  an  anthrax  culture  (Ogata  and  Jasuhara) ; 
also  that  the  injection  of  the  blood  of  a  frog  or  of  a  rat 
into  a  susceptible  animal  which  has  been  inoculated  with 
a  virulent  culture  of  the  anthrax  bacillus  will  restrain  the 
development  of  the  pathogenic  bacillus  and  prevent  the 
death  of  the  inoculated  animal. 

Buchner  (1889)  first  j)roved  by  experiment  that  the 
germicidal  power  of  the  blood  of  dogs  and  rabbits  does 
not  depend  upon  the  jiresence  of  the  cellular  elements, 
but  is  present  in  clear  serum  which  has  been  allowed  to 
separate  from  the  clot  in  a  cool  place.  Exposure  for  an 
hour  to  a  temperature  of  55°  C.  destroys  the  germicidal 
action  of  serum  as  well  as  of  blood  ;  the  same  efiect  is 


NATURAL   IMMUNITY.  23 

produced  by  heating  to  52°  C.  for  six  hours,  or  to  45.G'^  C. 
for  twenty  hours.  The  germicidal  power  of  blood-serum 
is  not  destro^^ed  by  freezing-  and  thawing,  but  is  lost  after 
it  has  been  kept  for  some  time  at  ordinary  tempera- 
tures. 

The  researches  of  Buchner,  of  Hankin,  and  others,  show 
that  this  germicidal  power  of  fresh  blood-serum  depends 
upon  the  presence  of  proteids,  to  which  the  first-named 
bacteriologist  has  given  the  name  of  "  alexins."  Hankin, 
in  his  paper  upon  the  origin  of  these  "  defensive  pro- 
teids "  in  the  animal  body  (1892),  arrives  at  the  conclusion 
that  while  they  are  present  in  the  cell-free  serum  they 
are  the  product  of  certain  leucocytes — Ehrlich's  eosino- 
phil cells.  He  believes  that  the  eosinophil  granules 
become  dissolved  in  the  serum  and  constitute  the  germi- 
cidal proteid  which  is  shown  to  be  present  by  experi- 
ments upon  bacteria.  According  to  Hankin  the  separ- 
ation of  these  granules  can  be  witnessed  under  the 
microscope.  They  first  accumulate  upon  one  side  of  the 
cell  and  then  gradually  disappear,  and  as  this  occurs  a 
considerable  increase  in  the  bactericidal  power  of  the 
serum  can  lie  demonstrated.  The  germicidal  power  of 
the  blood-serum  is  also  said  to  be  increased  when  the 
number  of  leucocytes  is  considerably  augmented,  as  oc- 
curs when  a  sterilized  culture  of  Vibrio  Metschnikovi  is 
injected  subcutaneously.  Also  by  treatment  which  favors 
a  separation  of  the  alexin  from  the  leucocytes,  i.e.,  a  so- 
lution of  the  eosinophil  granules.  This  may  be  accom- 
plished by  the  injection  of  an  extract  of  the  thymus  gland 
of  the  calf,  or  by  simply  alloAving  the  drawn  blood  to 
stand  for  several  hours  at  a  temperature  of  38''  to  40"  C. 

Buchner's  latest  communication  upon  the  subject  shows 
that   he   also    attributes  the    origin   of  the    germicidal 


24  IMMUNITY    AND    SEItUM-TIIEILVPY. 

proteid  in  fresh  blood-serum  to  the  leucocytes.  In  his 
paper  on  "  Immunity,"  read  at  the  Eighth  International 
Congress  on  Hygiene  and  Demography  (Budapest,  1894) 
he  calls  attention  in  the  first  place  to  the  fact  that  a 
clearly  marked  distinction  must  be  made  between  natu- 
ral immunity  and  acquired  immunity,  inasmuch  as  the 
"  alexins  "  and  "  antitoxins  "  have  "very  different  prop- 
erties. The  first-mentioned  proteids  are  destroyed  by  a 
comparatively  low  temperature  (55°  to  60°  C.)  while  the 
antitoxins  resist  a  considerably  higher  temperature,  and, 
unlike  the  alexins,  have  no  bactericidal  or  globulicidal 
action.  A  very  remarkable  fact  developed  in  Buchner's 
experiments  is  that  the  blood- serum  from  the  dog  and 
from  the  rabbit,  when  mixed,  neutralize  each  other  so  far 
as  their  germicidal  power  is  concerned. 

By  injecting  sterilized  emulsions  of  wheat-flour  paste 
in- the  pleural  cavity  of  rabbits  and  dogs  Buchner  suc- 
ceeded in  obtaining  an  exudate  which  had  more  decided 
germicidal  power  than  the  blood  or  serum  of  the  same 
animal.  This  was  evidently  due  to  the  large  number  of 
leucocytes  present,  but  not  to  their  phagocytic  action,  as 
was  shown  by  experiment.  By  freezing  the  exudate  the 
leucocytes  were  killed,  but  the  germicidal  action  of  the 
fluid  was  rather  increased  than  diminished  by  freezing. 
While  freezing  had  no  efi"ect  upon  the  germicidal  action 
of  the  pleural  exudate  this  was  always  neutralized  by 
exposure  to  a  temperature  of  55°  C.  The  observations 
referred  to  lead  Buchner  to  the  conclusion,  which  we  con- 
cur in,  that  phagocytosis  plays  an  entirely  subordinate 
role  in  the  germicidal  action  of  freshly  drawn  blood,  but 
that  this  action  does  depend,  to  a  considerable  extent  at 
least,  vxpon  the  leucocytes,  inasmuch  as  the  soluble  pro- 
teid to  which  it  is  due  has  its  origin  from  these  proto- 


NATURAL   IMMUNITY.  25 

plasmic  masses.    Bucliner  says  in  his  recent  paper,  above 
referred  to : 

"  Upon  the  basis  of  tbe  results  readied  a  reconciliation 
with  the  theory  of  phagocytosis  seems  very  possible,  as 
the  researches  upon  the  bactericidal  action  of  blood  and 
serum  lead  to  the  final  conclusion  that  the  leucocytes  are 
the  bearers  of  the  bactericidal  material.  The  extended 
facts  of  observation  which  Metschnikoff  and  his  associ- 
ates have  collected  in  favor  of  phagocytic  action  remain 
undisturbed.  But  the  explanation  of  the  process,  the 
conception  of  the  causal  connection,  must  be  somewhat 
different.  Through  sucli  a  conception  the  observations 
opposed  to  the  theory  of  phagocytosis  will  also  find  their 
explanation.  Tlius  in  the  observations  of  Eibbert,  which 
led  to  the  statement  of  his  wall-forming  theory,  the 
'  mantel '  of  leucocytes  which  surrounds  the  infected 
focus  may  very  well  represent  a  protecting  wall  which 
acts,  not  by  phagocytosis  but  through  the  germicidal 
proteid  given  off  by  the  leucocytes.  Also  in  streptococ- 
cus infection  recovery  often  occurs  without  any  evidence 
of  phagocytosis.  Miiller,  in  recent  experiments  on  an- 
thrax in  rats,  has  failed  entirely  to  observe  phagocytosis, 
although  the  organism  of  the  rat  is  very  unfavorable  for 
anthrax  infection.  Also  in  relapsing  fever  Tictin  was  not 
able  to  discover  any  phagocytosis  in  apea  from  which  the 
spleen  had  been  removed,  either  during  the  attack  or 
after  recovery,  when  this  occurred.  And  E.  Pfeiffer  re- 
cently demonstrated,  in  his  interesting  researches  upon 
the  killing  of  cholera  vibrios  in  the  peritoneal  cavity  of 
immunized  guinea-pigs,  that  their  rapid  destruction  oc- 
curred without  phagocytosis.  All  of  these  cases  present 
no  difficulty  as  soon  as  the  idea  is  abandoned  that  the  act 
of  devouring  ('  Auffressen  ')  constitutes  a  conditio  sine  qua 
non  for  the  bactericidal  activity  of  the  leucocytes." 

Emmerich,  Tsuboi,  Steinmetz,  and  Low  (1892),  as  a  re- 
sult of  extended  experiments,  arrived  at  the  conclusion 
that   the    germicidal    action    of  blood-serum    "  depends 


26  IMMUNITY   AND   SERUM-THERAPY. 

upon  a  specific  propei*ty  of  the  alkali-serum-albumin,  and 
that  it  is  a  purely  chemical  process."  The}^  state  that 
when  the  germicidal  power  is  neutralized  by  heat  it 
may  be  restored  by  the  addition  of  an  alkali.  Buchner 
repeated  the  experiments  of  Emmerich  and  his  associates 
and  obtained  similar  results,  but  interprets  them  differ- 
ently. According"  to  him  the  serum  does  not  regain  its 
germicidal  power,  but  after  the  addition  of  an  alkali  and 
subsequent  dialyzing  the  nutritive  value  of  the  serum  is 
so  diminished  that  the  bacteria  do  not  develop  in  it. 

The  failure  of  the  anthrax  bacillus  to  develop  in  inocu- 
lated white  rats  has  been  ascribed  by  Behriug  (1888)  to 
the  highly  alkaline  reaction  of  the  blood  and  tissue  juices 
of  this  animal.  Hankin  (1891)  in  extended  experiments 
arrived  at  a  different  conclusion.  From  the  spleen  and 
blood-serum  of  rats  he  isolated  a  globulin  possessing 
germicidal  properties,  to  which  he  ascribes  the  power  of 
the  rat's  blood  to  destroy  anthrax  bacilli,  without,  how- 
ever, rejecting  the  view  that  the  excessive  alkalinity  of 
the  blood  of  this  animal  may  be  a  factor  in  producing 
this  result.  Pane  (1892)  has  made  experiments  which 
give  additional  weight  to  the  assumption  that  the  alka- 
linity of  the  blood  is  an  important  factor  in  accounting 
for  immunity.  He  states  that  carbonate  of  soda,  dissolved 
in  water,  in  the  proportion  of  1  to  3,000,  has  a  decided 
germicidal  action  upon  the  anthrax  bacillus,  equal  to  that 
of  the  blood-serum  of  the  rabbit.  And  that  when  rabbit 
serum  is  completely  neutralized  it  no  longer  has  any  in- 
jurious action  on  anthrax  bacilli. 

Zagari  and  Innocente  (1892)  also  arrived  at  the  conclu- 
sion that  the  diminished  resistance  to  anthrax  infection 
resulting  from  curare-poisoning  in  frogs,  and  from 
chloral  or  alcohol  in  dogs  (Platania),  in  fowls  as  a  result 


NATURAL   IM3IUNITY.  27 

of  starvation  (Canalis  and  Morpurgo),  in  white  mice  as  a 
result  of  fatigue  (Cliarin  and  Roger),  is,  in  fact,  due  to  di- 
minished alkalinit}^  of  the  blood,  which  they  found  to  cor- 
respond wdtli  the  increased  susceptibility  resulting  from 
the  causes  mentioned. 

Buchner  (1892)  states  that  several  of  the  ammonium 
salts,  and  es^Decially  ammonium  sulphate,  cause  an  in- 
crease in  the  germicidal  action  of  blood-serum,  and  also 
increase  its  resistance  to  the  neutralizing  effects  of  heat. 
The  experiments  of  Pansini  and  Calabrese  (1894)  show, 
on  the  contrary,  that  the  addition  of  uric  acid  to  blood- 
serum  diminishes  its  bactericidal  activity,  as  does  also 
the  presence  of  glucose.  That  certain  infectious  dis- 
eases are  especially  virulent  in  persons  suffering  from 
diabetes  is  a  frequentlj"  repeated  clinical  observation. 

Van  Fodor  has  shown  by  experiment  that  the  injection 
of  an  alkali  into  the  circulation  of  a  rabbit  increases  its 
resistance  to  anthrax  infection  and  the  germicidal  activ- 
ity of  its  blood-serum.  The  same  bacteriologist  has 
found  that  when  a  rabbit  is  infected  with  anthrax,  the  al- 
kalinity of  its  blood  is  notably  increased  during  the  first 
twenty-four  hours,  when  we  may  suppose  that  the  pow- 
ers of  nature  are  brought  to  bear  to  resist  the  invading 
parasite,  and  that  after  this  time  it  rapidly  diminishes. 
Ten  hours  after  infection  (by  subcutaneous  inoculation?) 
the  alkalinity  of  the  blood  had  increased  21.5  per  cent. 
Shortly  before  the  death  of  the  animal  a  diminution  of 
26.3  per  cent,  was  noted.  This  diminution  w^as  observed 
in  thirty-four  out  of  thirty-nine  animals  experimented 
upon,  and  these  animals  succumbed  to  the  anthrax  infec- 
tion in  a  shorter  time  than  did  the  other  five  in  which 
there  was  no  such  diminution. 

It   seems  probable   that    the   germicidal   property   of 


28  IMMUNITY    AND   SERUM-THEKAPY. 

freshly  drawn  blood-serum  is  not  due  to  its  alkalinity, 
per  sc,  but  to  the  fact  that  the  germicidal  constituent  is 
only  soluble  in  an  alkaline  fluid.  The  recent  researches 
of  Vaug-hn,  McClintock,  and  Nov}^  indicate  that  this 
germicidal  constituent  is  a  nuclein.  Dr.  Vaughn  in  his 
last  iDublished  paper  upon  "  Nucleins  and  Nuclein  The- 
rapy," says  :  "  Kossel,  of  Berlin,  has  confirmed  our  state- 
ments concerning  the  germicidal  action  of  the  nucleins. 
Dr.  McClintock  and  I  have  also  demonstrated  that  the 
germicidal  constituent  of  blood-serum  is  a  nuclein. 
This  nuclein  is  undoubtedly  furnished  by  the  pol^^nu- 
clear  white  corpuscles."  Denys  has  recently  (1894)  re- 
ported the  results  of  experiments  made  in  his  laboratory 
by  Yan  der  Velde,  which  give  support  to  the  conclusion 
reached  by  Vaughn.  In  these  experiments  a  sterilized 
culture  of  staphylococci  was  injected  into  the  pleural 
cavity  of  rabbits  in  order  to  obtain  an  exudate.  At  in- 
tervals of  two  hours  this  exudate  was  obtained  by  killing 
one  of  the  animals  in  the  series  experimented  upon,  and 
at  the  same  time  blood  from  the  animal  was  secured. 
Both  the  exudate  and  the  blood  was  placed  in  a  centrifu- 
gal machine,  in  order  to  obtain  a  serum  free  from  corpus- 
cular elements.  The  germicidal  activity  of  the  serum 
was  then  tested.  The  general  result  of  the  experiments 
was  to  show  that  the  longer  the  interval  after  the  injec- 
tion into  the  pleural  cavity,  the  more  potent  the  germi- 
cidal activity  of  the  exudate  became  ;  and  that  there  was 
no  corresponding  increase  in  the  activity  of  tlie  blood- 
serum  obtained  from  the  circulation.  At  the  end  of  ten 
or  twelve  hours,  the  serum  from  the  exudate  killed  all  of 
the  staphylococci  in  a  bouillon  culture  twenty  times  as 
great  in  quantity  as  the  germicidal  serum  used  in  the  ex- 
periment.    The   absence   of  any  increase  in  germicidal 


NATURAL   IMMUNITY.  29 

power  iu  tlie  blood-serum  taken  from  the  general  circu- 
lation shows  that  the  notable  increase  manifested  by  the 
exudate  was  due  to  local  causes  ;  and  as  a  matter  of  fact 
it  corresponded  with  an  increase  in  the  number  of  leuco- 
cytes as  found  in  the  pleural  exudate. 

Thus  it  will  be  seen  that  the  independent  researches  of 
Haukin,  of  Buchner,  of  Vaughn,  and  of  other  competent 
bacteriologists,  have  led  them  to  the  same  ultimate  re- 
sult so  far  as  the  origin  of  the  germicidal  constituent  of 
the  blood  is  concerned,  and  that  the  leucocytes  aj)pear  to 
play  an  important  role  in  the  protection  of  the  animal 
body  from  invasion  by  bacteria  (natural  immunity),  al- 
though the  method  by  which  this  is  accomplished  differs 
from  that  suggested  by  the  writer  in  1881,  and  since 
strongly  supported  by  Metschnikofi'  and  his  associates — 
"  phagocytosis." 

With  reference  to  the  physiological  and  chenjical  char- 
acters of  the  nucleins  I  quote  from  Vaughn's  paper, 
above  referred  to,  as  follows  : 

"  Phj^siologically  nucleins  may  be  said  to  form  the 
chief  chemical  constituent,  of  the  living  parts  of  cells. 
Speaking  broadly,  we  may  say  that  the  nuclein  is  that 
constituent  of  the  cell  by  virtue  of  which  this  histologic 
unit  grows,  develops,  and  reproduces  itself.  It  is  the 
function  of  the  nuclein  of  the  cell  to  utilize  the  pabidum 
within  its  reach.  It  must  be  evident  that  those  tissues 
most  abounding  in  celhdar  elements  contain  relatively 
the  largest  amounts  of  nuclein.  It  must  also  be  seen  that 
it  is  by  virtue  of  their  nuclein  that  the  cells  of  "various 
organs  and  organisms  possess  and  manifest  their  indi- 
vidual peculiarities.  We  should  therefore  expect  to  find 
that  the  nuclein  of  the  yeast-cell  is  not  identical  with 
that  of  the  bacillus  tuberculosis,  and  that  the  nuclein  of 
the  spleen  differs  from  that  of  the  thj^roid  gland.  The 
number  of  kinds  of  nuclein  is  limited  only  by  the  varie- 


30  IMMUNITY   AND   SERU^I-TlIEKAPy. 

ties  of  cells.  Nuclein  is  the  chemical  basis  of  that  part 
of  the  cell  designatecl  by  the  histologist  as  the  nucleus, 
sometimes  called  chromatin  on  account  of  the  readiness 
with  which  it  absorbs  and  holds  coloring  agents.  It  is 
the  nuclein  of  the  bacterium  which  takes  up  and  retains 
the  stains,  and  it  is  on  account  of  the  fact  that  the  nuclein 
of  the  bacillus  tuberculosis  differs  from  that  of  other 
bacilli  that  we  are  able  to  distinguish  the  former  from 
the  latter  by  its  tinctorial  properties.  Differences  in  re- 
action with  staining  reagents,  so  plainly  seen  under  the 
microscope,  are  only  outward  manifestations  of  less  ap- 
jiareut  and  more  important  differences  in  chemical  com- 
position. 

"  Chemically  the  nucleins  are  complex,  proteid  bodies, 
characterized  especially  by  the  large  amount  of  phos- 
phorus which  they  contain.  The  phosphorus  exists  in  the 
form  of  nucleiuic  acid,  which  is  combined  with  a  highly 
complex  basic  substance.  So  far  as  we  know  at  present, 
the  nucleinic  acid  of  all  nucleins  is  the  same,  yet  the 
basic  part  differs  in  the  various  nucleins.  This  basic 
substance  yields,  as  decomposition  products,  one  or  more 
of  the  so-called  xanthin  bodies ;  adeuin,  guanin,  sarkin, 
and  xanthin.  Some  nucleins  yield  only  adenin,  and  these 
may  be  designated  as  adenyl  nucleinic  acids.  Those 
which  furnish  xanthin  most  abundantly  may  be  called 
xanthyl  nucleinic  acids.  Generally  ^pQaking,  the  nucleins 
are  insohible  in  dilute  acids  and  soluble  in  dilute  alkalies. 
They  resist  peptic  digestion  and  in  this  way  maj^  be  se]i- 
arated  from  most  other  proteid  bodies." 

It  has  been  shown  by  several  investigators  that  the 
number  of  leucocytes  increases  in  certain  infectious  dis- 
eases, and  this  increase,  together  with  an  increased  alka- 
linity of  the  blood,  which  has  heretofore  been  referred  to, 
appears  to  be  a  provision  of  nature  for  overcoming  the 
infection  which  has  already  occurred.     Billings,*  in  a  re- 


*  J.  S.  Billings,  Jr. :  The  Leucocytes  in  Croupous  Pneumonia,  Bulletin  of 
the  Johns  Hopkins  Hospital,  November,  1894,  p.  105. 


NATURAL   IMMUNITY.  31 

cent  paper,  gives  the  evidence  as  regards  croupous  pneu- 
monia.    Some  of  his  conclusions  are  given  below  : 

"  In  cases  of  pneumonia  pursuing  a  favorable  course 
there  is,  as  a  rule,  a  marked  increase  in  the  number  of 
leucocytes  during  the  febrile  iDeriod  of  the  disease.  This 
leucocytosis  is  probably  present  at  the  time  of  the  chill, 
and  may  be  very  marked  within  a  few  hours." 

"  In  cases  showing  extensive  involvement  of  both 
lungs  the  leucocytes  are  apt  tp  reach  a  higher  point  than 
in  those  cases  where  the  involvement  is  only  moderate." 

"  The  fatal  cases  may  show  either  the  ijresence  or  ab- 
sence of  leucocytosis.  In  those  cases  showing  a  leucocy- 
tosis, some  other  cause  of  death  than  the  virulence  of  the 
bacterial  poison  must  be  sought  for." 

"  The  prognosis  in  cases  sliowing  a  complete  and  con- 
tinuous absence  of  leucocytosis  is  unfavorable  as  a  rule." 

"  The  leucocytosis  in  pneumonia  is  a  so-called  pure 
leucocytosis,  i.e.,  an  increase  in  the  poly  nuclear  elements 
solely." 

The  experimental  evidence  submitted,  considered  in 
connection  with  the  extensive  literature  relating  to  "  pha- 
gocytosis," leads  us  to  the  conclusion  that  natural  immu- 
nity is  due  to  a  germicidal  substance  present  in  the  blood- 
serum  which  has  its  origin  (chiefly,  at  least)  in  the  leu- 
cocytes and  is  soluble  only  in  an  alkaline  medium.  And 
that  local  infection  is  usually  resisted  by  an  afilux  of  leu- 
cocytes to  the  point  of  invasion  ;  but  that  phagocytosis 
is  a  factor  of  secoudary  importance  in  resisting  parasitic 
invasion.  Also  that  general  infection,  at  least  in  some 
infectious  diseases,  is  resisted,  and  in  non-fatal  cases  over- 
come, by  an  increase  in  the  number  of  leucocytes  and  in 
the  alkalinit^^  of  the  blood-serum — which  favors  solution 
of  the  germicidal  proteids  contained  in  the  polynuclear 
leucocytes. 


32  IMMUNITY    AND    SEUUM-TIIEHAPY. 

This  conclusion  is  in  accord  with  certain  other  facts 
which  may  be  briefly  referred  to.  Numerous  experi- 
ments show  that  natural  immunity  may  be  overcome  by 
infection  with  an  excessive  number  of  pathogenic  bacte- 
ria and  their  products,  as  contained  in  a  culture ;  or  by 
an  unusually  virulent  variety.  According  to  Denys  the 
pathogenic  bacteria,  in  general,  resist  the  germicidal  ac- 
tion of  rabbit  serum  better  than  the  saprophytic  bacte- 
ria ;  and  of  two  varieties  of  the  same  pathogenic  species 
the  most  virulent  has  the  greater  resistance  to  this  ger- 
micidal action.  This  is  in  accord  witli  the  fact  that  nat- 
ural immunity  protects  completely  from  invasion  by  the 
ordinary  saprophytes,  by  which  man  and  the  lower  ani- 
mals are  surrounded  on  all  sides.  It  is  only  when  the 
circulation  is  arrested  in  the  entire  body,  or  in  a  portion 
of  it,  that  the  putrefactive  bacteria  succeed  in  invading 
the  tissues. 

The  action  of  the  germicidal  substance  present  in 
blood-serum  upon  bacteria  appears  to  be  a  quantitive 
one  so  far  as  the  bacteria  are  concerned,  and  it  may  also 
be  neutralized  by  their  products.  Thus  Bastin  (1892) 
concludes  from  his  experiments  that  the  germicidal  ac- 
tion of  blood -serum  from  the  dog  is  neutralized  or 
greatly  diminished  by  the  intravenous  injection  of  cult- 
ures of  bacteria  (S.  pyogenes  aureus,  Bac.  lactis  ?ero- 
genes)  in  considerable  amounts,  and  that  this  occurs  as 
well  when  the  bacteria  in  the  cultures  have  been  killed 
l)y  heat  as  when  they  are  living.  This  effect  is  mani- 
fested Avithin  two  minutes  after  the  injection,  and  is  said 
to  reach  its  maximum  inside  of  ten  minutes  ;  at  the  same 
time  there  is  a  diminution  in  the  coagulability  of  the 
blood.  At  the  end  of  five  or  six  hours  the  normal  coagu- 
lability and  germicidal  action  has  been  recovered.     The 


NATURAL   IMMUNITY.  33 

same  is  true  as  regards  freshly  drawn  blood,  hi  vitro. 
When  bacteria  or  their  products  are  added  in  excess  the 
germicidal  action  is  no  longer  manifested,  but  multi- 
plication occurs  at  once,  as  in  a  favorable  culture-fluid. 
Thus  Nissen  found  that  a  limited  number  of  cholera 
spirilla  added  to  freshly  drawn  rabbit's  blood  perished 
in  from  ten  to  forty  minutes ;  but  when  the  number  ex- 
ceeded 1,200,000  per  cubic  centimetre  an  increase  oc- 
curred at  the  end  of  five  hours. 

It  has  been  demonstrated  by  several  experimenters 
that  other  albuminous  fluids  possess  a  germicidal  power 
similar  to  that  manifested  by  freshly  drawn  blood.  Thus 
Nuttall  found  that  a  pleuritic  exudate  from  man  de- 
stroyed the  anthrax  bacillus  in  an  hour,  the  aqueous 
humor  of  a  rabbit  in  two  hours.  Prudden  found  that  the 
albuminous  fluid  obtained  from  a  hydrocele  sac,  or  from 
the  abdominal  cavity  in  ascites,  possesses  similar  germi- 
cidal power,  and  Fokker  has  demonstrated  that  fresh 
milk  destroys  the  vitality  of  certain  bacteria. 

Hankin  has  extracted  from  the  spleen  and  lymphatic 
ylands  of  dogs  and  cats  a  "  cell'  globulin  "  which  pos- 
sesses germicidal  power  similar  to  that  of  blood-serum. 
This  he  obtained  by  treatment  with  a  solution  of  sodi- 
um chloride  and  subsequent  precipitation  with  alcohol. 
This  "  globulin  "  is  insoluble  in  water  or  alcohol,  and 
does  not  dialyze. 


n. 

ACQUIRED  IMMUNITY. 

It  has  long  been  known  that,  in  a  considerable  number 
of  infectious  diseases,  a  single  attack,  however  mild,  af- 
fords protection  against  subsequent  attacks  of  the  same 
disease  ;  that  in  some  cases  this  protection  appears  to  be 
permanent,  lasting  during  the  life  of  the  individual ;  that 
in  others  it  is  more  or  less  temporary,  as  shown  by  the 
occurrence  of  a  subsequent  attack. 

The  protection  afforded  by  a  single  attack  not  only 
differs  in  different  diseases,  but  in  the  same  disease  varies 
greatly  in  different  individuals.  Thus  certain  individu- 
als have  been  known  to  suffer  several  attacks  of  small-pox 
or  of  scarlet  fever,  although,  as  a  rule,  a  single  attack  is 
protective.  Exceptional  susceptibility  or  insusceptibil- 
ity may  be  not  only  an  individual  but  a  family  character- 
istic, or  it  may  belong  to  a  particular  race. 

In  those  diseases  in  which  second  attacks  are  not  in- 
frequent, as,  for  exami^le,  in  i)neumonia,  in  influenza,  or 
in  Asiatic  cholera,  it  is  difficult  to  judge  from  clinical  ex- 
perience whether  a  first  attack  exerts  any  protective  in- 
fluence. But  from  experiments  upon  the  lower  animals 
we  are  led  to  believe  that  a  certain  degree  of  immunity, 
lasting  for  a  longer  or  shorter  time,  is  afforded  by  an 
attack  of  pneumonia  or  of  cholera,  and  probably  of  all 
infectious  diseases  due  to  bacterial  parasites.  In  the 
malarial  fevers,  which  are  due  to  a  parasite  of  a  different 


ACQUIRED   IMMUNITY.  36 

class,  one  attack  affords  no  protection,  but  rather  j)redis- 
poses  to  a  subsequent  attack. 

In  those  diseases  in  which  a  single  attack  is  generally 
recognized  as  being  protective  exceptional  cases  occur  in 
which  subsequent  attacks  are  developed  as  a  result  of 
unusual  susceptibility  or  exposure  under  circumstances 
especially  favorable  to  infection.  Maiselis  has  recently 
(1894)  gone  through  the  literature  accessible  to  him  for 
the  purpose  of  determining  the  frequency  with  which 
second  attacks  occur  in  the  various  diseases  below  men- 
tioned.    The  result  is  as  follows : 


Small-pox 

Scarlet  fever 

Measles 

Typhoid  fever 

Cholera 

These  figures  support  the  view  generally  entertained 
by  physicians  that  second  attacks  of  scarlet  fever  and  of 
measles  are  comparatively  rare,  while  second  attacks  of 
small-pox  are  not  infrequently  observed.  Considering 
the  very  large  number  of  cases  of  typhoid  fever  which 
occur  annually  in  all  parts  of  Europe  and  America  the 
number  of  second  attacks  collected  does  not  bear  a  very 
large  proportion  to  the  total  number  taken  sick,  although 
the  recorded  cases,  of  course,  fall  far  short  of  the  total 
number  of  second  attacks  of  this  and  the  other  diseases 
mentioned. 

The  second  attacks  of  cholera  recorded  are  not  numer- 
ous, and,  no  doubt,  a  carefully  conducted  investigation 
made  in  the  areas  of  endemic  prevalence  of  this  disease 
would  show  that  second  attacks  are  more  common  than 
is  indicated  by  these  figures. 


Second 
Attacks. 

Third 
Attacks. 

Fonrth 

Attacks. 

Total 

505 

9 

0 

514 

29 

4 

0 

33 

36 

1 

0 

37 

202 

5 

1 

208 

29 

3 

2 

34 

36  IMMUNITY  AND  SERUM-THERAPY. 

The  experimental  evidence  relating  to  protective  in- 
oculations in  infectious  diseases  dates  from  the  discovery 
by  Jenner  (1768)  of  the  protection  afforded  against  small- 
pox by  vaccination  with  lymph  taken  fi'om  the  vesicles 
of  cow-pox. 

To  Pasteur  must  be  accorded  the  credit  of  having  first 
shown  by  the  experimental  method  that  animals  may  be 
made  immune  against  other  infectious  diseases  than  the 
one  mentioned  by  inoculations  with  an  "  attenuated  vi- 
rus." Commencing-  with  his  experiments  upon  chicken 
cholera,  in  1880,  we  shall  briefly  trace  the  development 
of  our  knowledge  up  to  the  present  date. 

Having  demonstrated  that  the  disease  of  fowls  known 
as  chicken  cholera  is  due  to  a  sjiecific  microorganism, 
which  he  was  able  to  cultivate  in  artificial  media,  Pas- 
teur discovered  that  his  cultures  became  "  attenuated  "  as 
to  their  pathogenic  power  when  they  had  been  kept  for 
some  time  in  the  laborator}",  and  that  fowls  inoculated 
with  these  attenuated  cultures  suffered  a  comparatively 
mild  and  non-fatal  attack  of  the  disease,  and  were  subse- 
quently immune  against  the  pathogenic  action  of  the 
most  virulent  cultures,  or  against  contracting  the  disease 
by  contact  with  other  fowls  sufi'ering  from  it. 

Pasteur  at  once  comprehended  the  importance  of  this 
discovery,  and  inferred  that  what  was  time  of  one  infec- 
tious germ  -  disease  was  likely  to  be  true  of  others. 
Subsequent  researches,  by  this  savant,  and  by  other  bac- 
teriologists, have  justified  this  anticipation ;  and  the 
experimental  demonstration  has  been  made  in  a  consider- 
able number  of  similar  diseases. 

Pasteur  first  obtained  an  attenuated  virus  for  his  pro- 
tective inoculations  against  chicken  cholera  by  keeping 
his  cultures  for  a  considerable  time  freely  exposed  to  the 


ACQUIKEI)   IMMUNITY.  37 

air,  and  ascribed  the  attenuation  to  the  action  of  atmos- 
pheric oxygen.  He  found  that  when  cultures  were  made 
from  the  blopd  of  fowls  which  died  from  a  chronic  form 
of  the  disease,  they  possessed  an  exceptional  virulence 
which  was  not  lost  when  the  cultures  were  renewed  at 
short  intervals ;  but  that  by  keeping-  these  cultures  for 
two  months  the  virulence  was  greatly  diminished,  and 
fowls  usually  recovered  when  inoculated  with  such  cult- 
ures. When  kept  still  longer,  his  cultures  finally  lost 
all  pathogenic  power.  In  subsequent  exiDeriments  with 
the  bacillus  of  anthrax,  Pasteur  found  that  the  spores  of 
this  bacillus  retain  their  virulence  for  years,  and  that  it 
was  necessary  to  exclude  them  from  cultures  which  were 
to  serve  for  protective  inoculations.  When  cultivated  at 
a  temperature  of  42°  to  43°  C,  this  bacillus  does  not  form 
spores ;  and  Pasteur  ascertained  that  cultures  kept  at 
this  temperature  for  eight  days  no  longer  killed  suscep- 
tible animals,  and  could  be  used  in  his  protective  inocu- 
lations. 

Other  methods  of  attenuating  the  virulence  of  patho- 
genic bacteria  have  since  been  discovered.  Thus,  Tous- 
saint  has  shown  that  exposure  for  a  short  time  to  a  tem- 
perature a  little  below  that  which  destroys  the  vitality  of 
the  pathogenic  microorganism,  modifies  the  virulence  of 
a  culture  so  that  it  may  serve  for  protective  inoculations. 
In  the  case  of  Bacillus  anthracis,  Chauveau  has  shown 
that  a  temperature  of  50°  C,  maintained  for  eighteen 
minutes,  answers  the  i3urpose. 

Attenuation  of  virulence  may  also  be  effected  by  expos- 
ure to  certain  antiseptic  agents.  This  was  first  ascer- 
tained by  the  writer  in  experiments  made  in  1881,  the 
object  of  which  was  to  determine  the  comparative  value 
of  various  disinfecting  agents.    Incidentally  the  fact  was 


38  IMMUNITY  AISTD   SERUM-THERAPY. 

brought  out  that  agents  which  do  not  completely  destroy 
the  vitality  of  a  pathogenic  microorganism  may  cause  an 
attenuation  of  its  pathogenic  virulence.  In  the  experi- 
ments referred  to,  the  blood  of  a  rabbit  recently  dead 
from  a  form  of  seiDticoemia  induced  by  the  subcutaneous 
injection  of  my  own  saliva,  and  due  to  the  presence  of  a 
micrococcus  (Micrococcus  pneumoniae  crouposse),  was 
subjected  to  the  action  of  various  chemical  agents,  and 
subsequently  injected  into  a  rabbit  to  test  the  destruction 
of  virulence.  In  the  published  report  of  these  experi- 
ments the  following  statement  is  made : 

"  The  most  important  source  of  error,  however,  and  one 
which  should  be  kept  in  view  in  future  experiments,  is 
the  fact  that  a  protective  influence  has  been  shown  to 
result  from  the  injection  of  virus,  the  virulence  of  which 
has  been  modified,  without  being  entirely  destroyed,  by 
the  agent  used  as  a  disinfectant. 

"  Sodium  hyposulphite  and  alcohol  were  the  chemical 
reagents  which  produced  the  result  noted  in  these  expe- 
riments ;  but  it  seems  probable  that  a  variety  of  antisej)- 
tic  substances  will  be  found  to  be  equally  effective  when 
used  in  proper  proportion." 

Similar  results  have  since  been  reported  by  European 
bacteriologists.  Thus,  Chamberland  and  Roux  (1883) 
ascertained  that  the  anthrax  bacillus  could  be  attenuated 
by  adding  to  cultures  certain  antiseptic  agents — carbolic 
acid,  1  to  800  ;  bichromate  of  potash,  1  to  100. 

Another  method  of  attenuating  the  virus  of  a  patho- 
genic microorganism  is  that  recently  (1892)  discovered 
by  Brieger,  Kitasato,  and  Wassermann.  This  consists  in 
the  cultivation  of  pathogenic  bacteria  in  a  bouillon  made 
from  the  thymus  gland  of  a  calf.  It  was  found  that  the 
tetanus  bacillus  cultivated  in  this  bouillon  did  not  form 


ACQUIRED   IMMUNITY.  39 

spores,  and  had  comparatively  little  virulence.  Mice  or 
rabbits  inoculated  with  it  in  small  doses — 0.001  to  0.2  c.c. 
for  a  mouse — proved  to  be  subsequently  immune.  And 
the  blood-serum  of  an  immune  rabbit  injected  into  the 
peritoneal  cavity  of  a  mouse  (0.1  to  0.5  c.c.)  was  found  to 
g-ive  it  immunity  from  the  pathogenic  action  of  a  virulent 
culture  of  the  tetanus  bacillus.  Similar  results  were  ob- 
tained with  several  other  pathogenic  bacteria  cultivated 
in  the  thymus  bouillon — spirillum  of  cholera,  bacillus  of 
diphtheria,  typhoid  bacillus. 

Attenuation  of  virulence  may  also  be  effected  by  culti- 
vating the  anthrax  bacillus  in  the  body  of  a  non-suscepti- 
ble animal,  like  the  frog  (Lubarsch,  Petruschky)  ;  or  in 
the  blood  of  the  rat  (Behring) ;  by  exposure  to  sunlight 
(Arloing) ;  and  by  compressed  air  (Chauveau). 

It  is  a  matter  of  common  laboratory  experience  that 
many  pathogenic  bacteria  become  more  or  less  attenuated 
when  cultivated  for  a  considerable  time  in  artificial 
media,  even  when  the  cultures  are  renewed  at  short  inter- 
vals. This  is  true  of  the  micrococcus  of  pneumonia,  of 
streptococcus  pyogenes,  of  the  bacillus  of  diphtheria,  of 
the  spirillum  of  cholera,  and,  to  some  extent,  of  the  tu- 
bercle bacillus.  Indeed,  as  a  general  rule,  pathogenic 
bacteria  exhibit  greater  virulence  when  cultivated  in 
favorable  media,  and  when  recently  obtained  from  the 
body  of  a  susceptible  animal ;  and,  on  the  other  hand, 
pathogenic  virulence  is  diminished  by  cultivation  under 
unfavorable  conditions.  Probably  similar  circumstances 
produce  those  differences  in  the  type  of  epidemic  dis- 
eases, as  to  malignancy  or  comparative  mildness,  which 
have  been  frequently  noted  ;  external  conditions  unfavor- 
able to  the  development  of  the  specific  infectious  agent 
causing  an  attenuation  of  virulence,  and  the  reverse.    As 


40  IMMUNITY   AND   SERU3I-TIIERAPY. 

pathog'enic  virulence  depends,  to  a  considerable  extent  at 
least,  upon  tlie  formation  of  toxic  substances  during  the 
active  develoi^ment  of  the  pathogenic  micro-organism,  we 
infer  that  diminished  virulence  is  due  to  a  diminished 
production  of  these  toxic  substances. 

An  imi^ortant  step  was  made  in  the  progress  of  our 
knowledge  in  this  field  of  research  when  it  was  shown 
that  animals  may  be  made  immune  against  certain  infec- 
tious diseases  by  inoculating  them  with  filtered  cultures, 
containing  the  toxic  substances  just  refen-ed  to,  but  free 
from  the  living  bacteria  to  which  they  owe  their  origin. 
The  first  satisfactory  experimental  evidence  of  this  im- 
portant fact  was  obtained  by  Salmon  and  Smith  in  1886. 
These  bacteriologists  succeeded  in  producing  an  immu- 
nity in  pigeons  against  the  pathogenic  effects  of  the 
bacillus  of  hog  cholera,  which  is  very  fatal  to  these  birds, 
by  inoculating  them  with  sterilized  cultures  of  the  bacil- 
lus mentioned.  Similar  results  were  reported  by  Eoux, 
in  1888,  from  the  injection  into  susceptible  animals  of 
sterilized  cultures  of  the  anthrax  bacillus,  and  also  of  the 
bacillus  of  symptomatic  anthrax.  More  recently  (1890) 
Behring  and  Kitasato  have  shown  that  animals  may  be 
made  immune  against  the  pathogenic  action  of  the  bacil- 
lus of  tetanus  or  the  bacillus  of  diphtheria  bj^  the  injec- 
tion of  filtered,  germ-free  cultures  of  these  bacilli.  Sim- 
ilar results  have  been  obtained  by  G.  and  F.  Klemperer 
(1891),  in  experiments  upon  rabbits,  with  filtered  cultures 
of  the  micrococcus  of  croupous  pneumonia. 

In  Pasteur's  protective  inoculations  against  h3'dropho- 
bia  it  is  probable  that  the  immunity  which  is  developed 
after  infection  by  the  bite  of  a  rabid  animal  is  due  to  the 
toxin  (toxalbumin  ?)  of  this  disease  present  in  the  emul- 
sion of  spinal  cord  which  is  used  in  these  inoculations. 


ACQUIRED   IMMUNITY.  41 

There  is  also  some  evidence  to  show  that  a  certain  de- 
gree of  immunity  ag-ainst  tuberculosis  may  be  produced 
in  g-uinea-pig-s  by  injections  of  the  toxic  substances  de- 
veloped during  the  growth  of  the  tubercle  bacillus — 
Koch's  tuberculin. 

Evidently  the  facts  stated  have  an  important  bearing 
upon  the  rationale  of  acquired  immunity,  and  they  ap- 
pear to  support  the  explanation  offered  by  the  writer  in 
a  iiaper  published  in  The  American  Journal  of  the  Medical 
Sciences  in  1881,  namely,  that  immunity  depends  upon  an 
acquired  tolerance  to  the  toxic  products  of  pathogenic 
bacteria.     In  the  paper  referred  to  I  say  : 

"  This  explanation  is,  I  believe,  to  be  found  in  the  pecul- 
iar properties  of  the  protoplasm,  which  is  the  essential 
framework  of  every  living  organism.  The  properties  re- 
ferred to  are  :  The  tolerance  which  living  protoplasm  may 
acquire  to  certain  agents  Avliich,  in  the  first  instance, 
have  an  injurious  or  even  fatal  influence  upon  its  vital 
activity,  and  the  property  which  it  possesses  of  transmit- 
ting its  peculiar  qualities,  inherent  or  acquired,  through 
numerous  generations,  to  its  offshoots  or  progeny. 

"  There  can  be  but  little  doubt  that  protoplasm  is  the 
essential  living  portion  of  the  cellular  elements  of  ani- 
mal and  vegetable  tissues,  but  as  our  microscoj^ic  analy- 
sis of  the  tissues  has  not  gone  beyond  the  cells  of  which 
they  are  composed,  and  is  not  likely  to  reveal  to  us  the 
complicated  molecular  structure  of  the  protoplasm  upon 
which,  possibly,  the  properties  under  consideration  de- 
pend, it  will  be  best  for  the  present  purpose  to  limit  our- 
selves to  a  consideration  of  the  living  cells  of  the  body. 
These  cells  are  the  direct  descendants  of  pre-existing" 
cells,  and  may  all  be  traced  back  to  the  sperm-cell  and 
germ-cell  of  the  parents.  Now,  the  view  which  I  am  en- 
deavoring to  elucidate  is,  that  during  a  non-fatal  attack 
of  one  of  the  specific  diseases  the  cellular  elements  im- 
plicated which  do  not  succumb  to  the  destructive  influ- 


42  IMMUNITY   AND   SERUM-THERAPY. 

ence  of  the  poison,  acquire  a  tolerance  to  this  poison 
which  is  transmissible  to  their  proo-eny,  and  which  is  the 
reason  of  the  exemption  which  the  individual  enjoys  from 
future  attacks  of  the  same  disease. 

"  The  known  facts  in  regard  to  the  hereditary  transmis- 
sion, by  cells,  of  acquired  properties,  make  it  very  easy 
to  believe  in  the  transmission  of  such  a  tolerance  as  we 
imagine  to  be  acquired  during  the  attack,  and  if  it  is 
shown  by  analogy  that  there  is  nothing  improbable  in 
the  hypothesis  that  such  a  tolerance  is  acquired,  we 
shall  have  a  rational  explanation,  not  of  heredity  and  the 
mysterious  proi^erties  of  jjrotoplasm,  but  of  the  p^irticu- 
lar  result  under  consideration. 

"  The  transmission  of  acquired  properties  is  shown  in 
the  budding  and  grafting  of  choice  fruits  and  flowers, 
produced  by  cultivation,  upon  the  wild  stock  from  which 
they  originated.  The  acquired  properties  are  trans- 
mitted indefinitely,  and  the  same  sap  which  on  one  twig 
nourishes  a  sour  crab-apple,  on  another  one  of  the  same 
branch  is  elaborated  into  a  delicious  pippin. 

"Numerous  examples  in  illustration  of  the  same  fact 
may  be  drawn  from  the  animal  kingdom  ;  thus,  the  same 
mother  may  give  birth  to  two  children  by  different 
fathers ;  the  one  may  inherit  a  predisposition  to  con- 
sumption, and  the  other  to  insaniity  ;  and  this  inheri- 
tance, which  only  manifests  itself  at  the  end  of  many 
years,  has  been  transmitted  from  the  original  sperm-cells 
of  the  respective  fathers,  through  countless  generations 
of  cells  which  have  lived  and  died,  leaving-  their  progeny 
to  perform  their  functions. 

"The  immunity  which  an  individual  enjoys  from  any 
particular  disease  must  be  looked  upon  as  a  power  of 
resistance  possessed  by  the  cellular  elements  of  those 
tissues  of  his  body  which  would  yield  to  the  influence  of 
the  iDoison  in  the  case  of  an  unprotected  person.  There 
is  every  reason  to  believe  that  it  is  upon  the  living  por- 
tion of  the  tissues,  or  the  protoplasm  of  the  body,  that 
the  disease-poisons  act ;  for  if  it  were  upon  non-living 


ACQUIRED   IMMUNITY.  43 

matter — formed  material,  Beale — and  we  had  to  deal  only 
with  chemical  phenomena,  it  would  be  impossible  to  ac- 
count for  the  fact  that  like  causes  do  not  always  produce 
like  results.  On  the  other  hand,  the  resistance  of  living- 
matter  to  certain  destructive  influences  is  a  property 
dependent  upon  vitality.  Thus,  living-  protoplasm  resists 
the  action  of  the  bacteria  of  putrefaction,  while  dead  pro- 
toplasm quickly  underg-oes  putrefactive  changes.  Again, 
it  seems  probable  that  in  conditions  of  debility  from  age, , 
sickness,  starvation,  or  any  other  cause,  the  vital  resist- 
ing- power  of  the  protoplasm  is  reduced,  and  certain 
agents  which,  under  more  favorable  conditions,  would 
be  powerless  for  harm,  may  overcome  this  vital  resist- 
ance. 

"The  tolerance  to  narcotics,  opium,  tobacco,  etc.,  re- 
sulting from  a  gradual  increase  of  dose,  may  be  cited  as 
an  example  of  acquired  tolerance  by  living-  protoplasm 
to  poisons,  which  at  the  outset  would  have  been  fatal  in 
much  smaller  doses.  There  can  be  little  doubt  that  in 
this  instance  it  is  the  living  protoplasm  of  the  nervous 
tissues  upon  which  the  poison  acts  to  produce  its  char- 
acteristic effects. 

"  But  it  is  in  the  specific  diseases  in  which  a  single  at- 
tack proves  protective  that  I  find  the  best  proof  that  the 
cellular  elements  of  the  body  may  acquire  a  tolerance 
during  the  attack  which,  being  transmitted  to  their  cellu- 
lar progeny,  furnishes  the  protection  which  the  individ- 
ual enjoys. 

"Let  us  take  a  particular  case.  In  yellow  fever  the 
immediate  effect  of  the  poison  seems  to  be  to  arrest  vital 
processes  generally — nutrition,  secretion,  excretion — and 
in  fatal  cases  we  find  that  the  protoplasm  of  various  or- 
gans and  tissues  has  undergone  degenerative  changes ; 
this  is  especially  true  of  the  liver-cells.  Now,  we  have 
every  reason  to  believe  that  this  occurs  in  a  less  degree 
in  non -fatal  cases,  but  that  a  sufficient  number  of  cells 
having  resisted  the  destructive  influence  of  the  poison, 
and  become   accustomed  to  its  presence,  resume  their 


44  IMMUNITY    AND   SERUM-THERAPY. 

functions,  and  that  thus  the  vital  processes  upon  which 
the  life  of  the  individual  depends  are  again  carried  on  in 
the  very  presence  of  the  poison,  Avhich  at  first  paralyzed 
or  destroyed  the  vital  activity  of  certain  cells.  The  case 
is  more  striking-  in  small-pox,  in  which  there  is  an  un- 
doubted increase  of  the  poison  in  the  tissues  during  the 
progress  of  the  disease,  but  in  the  first-mentioned  disease 
the  patient  commonly  remains  during  his  sickness  in  the 
infected  atmosphere,  the  breathing  of  which  i3roduced 
the  attack  from  which  he  is  suifering. 

"  The  protection  from  yellow  fever  resulting  from  ac- 
climation— if,  indeed,  there  is  such  a  thing  as  acclimation 
independent  of  an  attack  of  the  disease — seems  'to  be  a 
tolerance  acquired  by  repeated  exposure  to  the  poison  in 
quantities  not  sufficient  to  produce  an  attack. 

"  The  tolerance  enjoyed  by  the  negro  race  to  the  mala- 
rial poison  is  probably  the  result  of  long  residence  in 
malarious  regions.  Natural  selection  has  doubtless 
come  into  play  here  in  establishing  this  tolerance  as  a 
race  peculiarity. 

"I  would,  then,  place  acclimation,  inoculation  by  at- 
tenuated viruses,  and  an  attack  of  any  one  of  the  specific 
diseases,  all  in  the  same  category,  so  far  as  the  explana- 
tion of  the  protection  afforded  is  concerned  ;  and,  accord- 
ing to  my  view,  the  explanation  of  this  phenomenon  is 
to  be  found  in  the  peculiar  properties  of  living  proto- 
plasm which  enable  it,  within  certain  limits,  to  adapt 
itself  to  varying  conditions  and  injurious  influences,  and 
to  transmit  the  impression  or  modification  received  in  so 
doing  to  its  offshoots,  which  continue  to  perform  its 
functions  during  the  life  of  the  individual." 

In  my  chapter  on  "  Bacteria  in  Infectious  Diseases,"  in 
"  Bacteria,"  published  in  the  spring  of  1884, 1  say  : 

"  It  may  be  that  the  true  explanation  of  the  immunity 
afforded  by  a  mild  attack  of  an  infectious  germ-disease 
is  to  be  found  in  an  acquired  tolerance  to  the  action  of  a 


ACQUIRED   IMMUNITY.  45 

chemical  poison  produced  by  the  microorg-anism,  and 
consequent  ability  to  bring  the  resources  of  nature  to 
bear  to  restrict  invasion  by  the  parasite." 

The  resources  of  nature  supposed  to  be  brought  to 
bear  in  restricting  invasion  by  the  parasite  are  referred 
to  on  i^age  15 ;  the  explanation  offered  being  the  same 
as  that  subsequently  known  as  the  Metchnikoff  theory. 

As  shown  in  the  discussion  of  natural  immunity,  the 
leucocytes  undoubtedly  play  an  important  part  in  the 
protection  of  the  individual  against  invasion  by  patho- 
genic bacteria.  The  experiments  of  Metchnikoff  and 
others  show  that  the  introduction  of  pathogenic  bacteria 
into  the  subcutaneous  tissues  of  an  immune  animal  often 
leads  to  an  emigration  of  leucocytes  to  the  jDoint  of  inva- 
sion far  exceeding  that  which  occurs  in  one  which  has 
not  a  natural  or  acquired  immunity  for  the  particular 
microorganism  thus  introduced.  This  fact,  however, 
does  not  demonstrate  the  truth  of  the  theory  of  phagocy- 
tosis, and,  as  we  shall  see,  recent  researches  indicate  that 
the  principal  factor  in  the  i^roduction  of  acquired  immu- 
nity is  the  presence  in  the  blood  of  the  immune  animal 
of  some  substance  capable  of  neutralizing  the  toxic  i^rod- 
ucts  of  the  particular  pathogenic  microorganism  against 
which  immunity  exists,  or  of  destroying  the  "germ" 
itself. 

These  substances  are  called  antitoxins.  As  pointed 
out  by  Buchner  in  a  recent  i^aper  ((>/?.  cit.,  p.  24),  the  an- 
titoxins differ  essentially  from  the  so-called  alexins,  to 
which  natural  immunity  is  ascribed.  The  alexins  are 
characterized  by  their  germicidal  and  globulicidal  action 
— they  destroy  both  the  red  corpuscles  and  the  leuco- 
cytes of  animals  belonging  to  a  different  species  from 
that  from  which  they  have  been  obtained,  and  by  their 


46  IMMUNITY   AND   SEEUM-THEKAPY. 

coagulability  and  instability — destroyed  by  sunlight  and 
by  a  temperature  of  50°  to  55°  C.  On  the  other  hand, 
the  antitoxins  best  known  (diphtheria  and  tetanus)  have 
no  germicidal  or  globulicidal  action ;  they  resist  the 
action  of  sunlight  and  require  a  temperature  of  70°  to 
80°  C.  for  their  destruction. 

Our  knowledge  of  the  antoxins  dates  from  the  experi- 
ments made  in  the  Hygienic  Institute  of  Tokio,  by  Ogata 
and  Jasuhara,  in  1890.  These  bacteriologists  discovered 
the  important  fact  that  the  blood  of  an  animal  immune 
against  anthrax  contains  some  substance  which  neutral- 
izes the  toxic  products  of  the  anthrax  bacillus.  When 
cultures  were  made  in  the  blood  of  dogs,  frogs,  or  of 
white  rats,  which  animals  have  a  natural  immunity 
against  anthrax,  they  were  found  not  to  kill  mice  inocu- 
lated with  them.  Further  experiments  showed  that  mice 
inoculated  with  virulent  anthrax  cultures  did  not  suc- 
cumb to  anthrax  septicaemia  if  they  received  at  the  same 
time  a  subcutaneous  injection  of  the  blood  of  an  immune 
animal.  Further,  it  was  found  that  mice  which  had  sur- 
vived anthrax  infection  as  a  result  of  this  treatment  were 
immune  at  a  later  date  (after  several  weeks),  when  inocu- 
lated with  a  virulent  culture  of  the  anthrax  bacillus.  In 
the  same  year  (1890)  Behring  and  Kitasato  discovered 
that  the  blood  of  an  animal  which  has  an  acquired  immu- 
nity against  tetanus  or  diphtheria,  when  added  to  a  viru- 
lent culture  of  one  or  the  other  of  these  bacilli,  neutral- 
izes the  pathogenic  power  of  such  cultures,  as  shown  by 
inoculation  into  susceptible  animals.  And  also  that  cult- 
ures from  which  the  bacilli  have  been  removed  by  filtra- 
tion, and  which  kill  susceptible  animals  in  very  small 
amounts,  have  their  toxic  potency  destroyed  by  adding 
to  them  the  blood  of  an  immune  animal,  which  is  thus 


ACQUIRED   IMMUNITY.  47 

directly  proved  to  contain  an  antitoxin— wliicli  compara- 
tive experiments  show  not  to  be  present  in  the  blood  of 
non-immune  animals.  In  the  experiments  of  Behriug- 
and  Kitasato  referred  to,  it  was  found  that  5  c.c.  of 
serum  from  the  blood  of  an  immune  rabbit,  mixed  with 
1  c.c'  of  a  virulent  filtrate  of  the  tetanus  bacillus,  and 
allowed  to  stand  for  twenty-four  hours,  completely  neu- 
tralized its  toxic  power,  as  shown  by  inoculations  in 
mice ;  0.2  c.c.  of  this  mixture  injected  into  a  mouse  Avas 
without  eiiect,  while  0.0001  c.c.  of  the  filtrate,  without 
such  admixture,  was  infallibly  fatal  to  mice.  The  mice 
inoculated  with  this  mixture  remained  immune  for  forty 
or  fifty  days,  after  which  they  gradually  lost  their  immu- 
nity. The  blood  or  serum  from  an  immune  rabbit,  when 
preserved  in  a  dark,  cool  place,  retained  its  power  of 
neutralizing  the  tetanus  toxalbumin  for  about  a  week, 
after  which  time  it  gradually  lost  this  power.  Behring 
and  Kitasato  have  also  shown  that  the  serum  of  a  diph- 
theria-immune rabbit  destroys  the  potent  toxalbumin  in 
diphtheria  cultures.  It  does  not,  however,  possess  any 
germicidal  power  against  the  diphtheria  bacillus. 

Tizzoni  and  Cattani  (1891)  have  obtained  similar  re- 
sults. By  repeated  inocidations  with  gradually  increas- 
ing doses  of  the  tetanus  toxin  they  succeeded  in  pro- 
ducing- immunity  in  dogs  and  other  animals  ;  and  they 
found  by  experiment  that  a  small  amount  of  blood- 
serum  from  an  immune  dog  completely  destroys  the 
toxic  power  of  a  filtrate  from  cultures  of  the  tetanus 
bacillus — one  or  two  drops  of  serum  neutralized  0.5  c.c. 
of  filtrate  after  fifteen  or  twenty  minutes'  contact.  They 
also  ascertained  that  small  amounts  of  blood-serum  from 
an  immune  dog  injected  into  Avliite  mice  produced  im- 
munity in  these  animals.     In  a  subsequent  paper,  pub- 


48  IMMUNITY    AND    SERUM-THERAPY. 

lislied  in  the  same  year  (1891),  the  authors  named  re- 
ported that  the  tetanus  antitoxin  in  blood-serum  from  an 
immune  dog-  is  destroyed  by  exposure  to  a  temperature 
of  68°  C.  for  half  an  hour,  or  by  contact  with  acids  and 
alkalies ;  also  that  it  does  not  pass  through  a  dialyzing- 
membrane, 

Kitasato  in  the  same  year  (1891)  published  his  impor- 
tant researches  upon  immunity  from  tetanus.  He  pro- 
duced an  immunity  in  rabbits,  which  lasted  about  two 
months,  by  inoculating-  them  with  the  filtrate  from  a 
culture  of  the  tetanus  bacillus,  and  subsequently,  in 
the  same  locality,  with  3  c.c.  of  a  one  per  cent,  solution 
of  terchloride  of  jodine ;  this  last  solution  was  injected 
subcutaneously  in  the  same  dose  at  intervals  of  twenty- 
four  hours  for  five  days.  Kitasato  also  ascertained  that 
a  small  quantity  of  blood  (0.2  c.c.)  from  an  immune  rab 
bit,  when  injected  into  the  abdominal  cavity  of  a  mouse., 
gave  it  immunity  from  the  effects  of  inoculations  with 
the  tetanus  bacillus.  He  also  made  the  important  dis- 
covery that  mice  which  were  inoculated  with  a  virulent 
culture  of  the  tetanus  bacillus,  and  which  subsequent- 
ly, after  tetanic  symptoms  had  apjieared,  received  in 
the  cavity  of  the  abdomen  an  injection  of  blood-serum 
from  an  immune  mouse,  were  preserved  from  death. 
The  power  of  the  blood  of  an  immune  animal  to  neutral- 
ize the  tetanus  poison  was  also  shown  by  mixing  the  fil- 
trate from  a  virulent  culture  with  blood-serum  from  an 
immune  animal,  and  allowing  it  to  stand  for  twenty -four 
hours ;  a  dose  three  hundred  times  greater  than  would 
have  sufficed  to  kill  a  mouse  proved  to  be  without  effect 
after  such  admixture  with  blood-serum.  Control  experi- 
ments showed  that  blood-serum  from  animals  not  im- 
mune had  no  effect  upon  the  virulence  of  the  filtrate  from 


ACQUIRED   IMMUNITY.  49 

tetanus  cultures.  Tlie  duration  of  immunity  induced  in 
this  way  was  found  to  be  from  forty  to  fifty  days. 

Yaillard  (1891)  lias  succeeded  in  producing  immunity 
in  rabbits  by  repeated  injections  into  the  circulation  of 
filtered  cultures  which  had  been  exposed  for  an  hour 
to  a  temperature  of  60°  C.  At  a  temperature  of  65°  C. 
both  the  toxic  and  the  immunizing-  action  of  the  filtrate 
were  destroyed. 

In  1891  G.  and  F.  Klemperer  published  an  important 
memoir,  in  which  they  give  an  account  of  their  re- 
searches relating  to  the  question  of  immunity,  etc.,  in 
animals  subject  to  the  form  of  septicaemia  produced  by 
the  micrococcus  of  croupous  pneumonia.  They  were  able 
to  produce  immunity  in  susceptible  animals  by  introduc- 
ing into  their  bodies  filtered  cultures  of  this  micrococcus, 
and  proved  by  experiment  that  this  immunity  had  a  du- 
ration of  at  least  six  months.  They  also  arrived  at  the 
conclusion  that  the  immunity  induced  by  injecting  fil- 
tered cultures  into  susceptible  animals  is  due  to  the  pro- 
duction of  an  antitoxin  in  the  body  of  the  animal. 

Emmerich,  at  the  meeting  of  the  International  Con- 
gress for  Hygiene  and  Demography,  in  London  (1891), 
reported  results  corresponding  with  those  obtained  by 
G.  and  F.  Klemperer  as  regards  the  i)roduction  of  im- 
munity. He  also  gave  an  account  of  experiments  by 
Donissen  in  which  the  injection  of  20  to  25  c.c.  of  blood 
or  expressed  tissue  juices,  filtered  through  porcelain, 
from  an  immune  rabbit  into  an  unprotected  rabbit,  sub- 
sequently to  infection  by  a  bouillon  culture  of  "  diplococ- 
cus  lineumonise,"  prevented  the  development  of  fatal 
septicaemia.  Similar  results  have  been  reported  by  Em- 
merich and  Fawitzky  in  experiments  made  upon  mice 
with  the  bacillus  of  hog  erysipelas  (rothlauf). 


50  IMMUNITY   AND   SERUM-THERAPY. 

Brieg-er,  Kitasato,  and  Wassermann  have  reported 
(1892)  their  success  in  conferring-  immunity  upon  guinea- 
pigs  against  the  pathogenic  action  of  the  cholera  spiril- 
him.  They  found  that  attenuated  cultures  suitable  for 
use  as  *'  vaccines  "  could  be  obtained  by  cultivating-  the 
spirillum  in  bouillon  made  from  the  thymus  gland  of  the 
calf,  by  which  means  they  have  also  obtained  attenuated 
cultures  of  the  bacillus  of  diphtheria,  the  bacillus  of 
typhoid  fever,  the  bacillus  of  tetanus,  and  the  strejjto- 
coccus  of  erysipelas.  Guinea-pigs  inoculated  with  a  cult- 
ure in  thymus  bouillon,  which  had  been  subjected  to  a 
temperature  of  65°  C.  for  fifteen  minutes,  were  found, 
after  twenty -four  hours,  to  be  immune  against  virulent 
cultures  in  twice  the  amount  which  would  otherwise  have 
been  fatal. 

During  the  jiast  two  or  three  years  numerous  addi- 
tional experiments  have  been  reported  which  confirm 
the  results  already  referred  to,  and  show  that  immunity 
may  be  i^roduced  in  a  similar  manner  against  the  toxic 
products  of  various  other  pathogenic  bacteria  —  the 
typhoid  bacillus,  the  "  colon  bacillus,"  streptococcus  pyo- 
genes, staphylococcus  pyogenes  aureus  and  albus,  etc. 

The  Italian  investigators  Tizzoni  and  Centanni,  in 
18&2,  published  a  jireliminary  communication  in  which 
they  gave  the  results  of  experiments  which  appear  to 
show  that  in  guinea-pigs  treated  with  tuberculin,  by 
Koch's  method,  a  substance  is  developed  which  neutral- 
izes the  pathogenic  potency  of  the  tubercle  bacillus. 
Professor  Tizzoni  and  his  associate.  Dr.  Schwarz,  have 
also  (1892)  obtained  evidence  that  there  is  an  antitoxin  of 
rabies.  Blood-serum  taken  from  a  rabbit  having  an  arti- 
ficial immunity  against  this  disease  was  found  to  neutral- 
ize in  vitro  the  virulence  of  the  spinal  marrow  of  a  rabid 


ACQUIRED   IMMUNITY.  61 

animal  after  a  contact  of  live  hours.  The  blood-serum  of 
dogs  having  an  acquired  immunity  against  rabies  was 
found  to  have  a  similar  action,  but  in  much  less  deg-ree. 
The  substance  (antitoxin)  present  in  the  blood-serum  of 
an  immune  rabbit  does  not  dialyze  ;  it  is  precipitated  by 
alcohol,  and  preserves  its  activity,  to  a  considerable  ex- 
tent, after  precij^itation ;  it  is  soluble  in  giycerin  and  is 
said  to  have  the  general  chai-acters  of  a  "  globulin."  The 
experimenters  named  also  succeeded  in  conferring  im- 
munity upon  susceptible  animals  by  injecting  into  them 
blood-serum  containing  this  antitoxin.  According  to  the 
Italian  investigators  named,  tlie  antitoxins  of  tetanus  and 
of  rabies  are  found  only  in  the  blood-serum  of  immune 
animals  and  not  in  the  tissues  (nervous  or  muscular),  or 
in  the  parenchyma  of  the  various  organs. 

Professor  Ehrlich,  of  Berlin,  in  1891,  published  the  re- 
sults of  some  researches  which  have  an  important  bear- 
ing upon  the  explanation  of  acquired  immunity,  and 
which  show  that  susceptible  animals  may  be  made  im- 
mune against  the  action  of  certain  toxic  proteids  of  vege- 
table origin,  other  than  those  produced  by  bacteria ;  also 
that  this  immunity  depends  upon  the  presence  of  an 
antitoxin  in  the  blood-serum  of  the  immune  animals. 

The  experiments  of  Ehrlich  were  made  with  two  very 
potent  toxalbumins,  one — ricin — from  the  castor-oil  bean, 
the  otluer — abrin — from  the  jequirity  bean.  The  toxic 
potency  of  ricin  is  somewhat  greater  than  that  of  abrin, 
and  it  is  estimated  by  Ehrlich  that  1  gm.  of  this  sub- 
stance would  suffice  to  kill  one  and  a  half  million  of 
guinea-pigs.  When  injected  beneath  the  skin  in  dilute 
solution  it  produces  intense  local  inflammation,  resulting 
in  necrosis.  Mice  are  less  susceptible  than  guinea-pigs, 
and  are  more  easily  made  immune.    This  is  most  readily 


52  IMMUNITY   AND   SERUM-THERAPY. 

accomplished  by  giving  them  small  and  graduallj^  in- 
creasing doses  with  their  food.  As  a  result  of  this  treat- 
ment the  animal  resists  subcutaneous  injections  of  200  to 
400  times  the  fatal  dose  for  animals  not  having  this  arti- 
ficial immunity.  The  fatal  dose  of  abrin  is  about  double 
that  of  ricin.  When  injected  into  mice  in  the  proportion 
of  1  c.c.  to  20  gm.  of  body-weight,  a  solution  of  1  i)art 
in  100,000  of  water  x)roved  to  be  a  fatal  dose.  The  local 
effects  are  also  less  pronounced  when  solutions  of  abrin 
are  used.  These  consist  principally  of  an  extensive  in- 
duration of  the  tissues  around  the  j^oint  of  injection,  and 
a  subsequent  falling  off  of  the  hair  over  the  indurated 
area.  When  introduced  into  the  conjunctival  sac,  how- 
ever, abrin  prodiices  a  local  inflamrnation  in  smaller 
amounts  than  ricin,  a  solution  of  1  to  800  being  sufficient 
to  cause  a  decided,  but  temporary,  conjunctivitis.  Solu- 
tions of  1  to  50,  or  1  to  100,  of  either  of  these  toxalbumins, 
introduced  into  the  eye  of  a  mouse,  gave  rise  to  a  pan- 
ophthalmitis which  commonly  resulted  in  destruction  of 
the  eye.  But  in  mice  which  have  been  rendered  im- 
mune, by  feeding  them  for  several  weeks  with  food  con- 
taining one  of  these  toxalbumins,  no  reaction  follows  the 
introduction  into  the  eye  of  the  strongest  possible  solu- 
tion, or  of  a  paste  made  by  adding  abrin  to  a  ten  per 
cent,  salt  solution.  Ehrlich  gives  the  following  explana- 
tion of  the  remarkable  degree  of  immunity  established  in 
his  experiments  by  the  method  mentioned  : 

"  All  of  these  phenomena  depend,  as  may  easily  be 
shown,  upon  the  fact  that  the  blood  contains  a  body — 
antiabrin — which  completely  neutralizes  the  action  of  the 
abrin,  probably  by  destroying  this  body." 

In  a  later  paper  (1892)  Ehrlich  has  given  an  account  of 
subsequent  experiments  which  show  that  the  young  of 


ACQUIRED   IMMUNITY.  53 

« 

mice  which  have  an  acquired  immunity  for  these  vege- 
table toxalbumins  may  acquire  immunity  from  the  inges- 
tion of  their  mother's  milk ;  and  also,  that  immunity 
from  tetanus  may  be  acquired  in  a  brief  time  by  young 
mice  through  their  mother's  milk.  In  his  tetanus  experi- 
ments Ehrlich  used  blood-serum  from  an  immune  horse 
to  give  immunity  to  the  mother-mouse,  when  her  young 
were  already  seventeen  days  old.  Of  this  blood-serum 
2  c.c.  was  injected  at  a  time  on  two  successive  days. 
The  day  after  the  injection  one  of  the  sucklings  received 
a  tetanus  inoculation,  by  means  of  a  splinter  of  wood  to 
which  spores  were  attached.  The  animal  remained  in 
good  health,  while  a  much  larger  control  mouse,  inocu- 
lated in  the  same  way,  died  of  tetanus  at  the  end  of  twenty- 
six  hours.  Other  sucklings,  inoculated  at  the  end  of 
forty-eight  hours  and  of  seventy-two  hours  after  the 
mother  had  received  the  injection  of  blood-serum,  like- 
wise remained  in  good  health,  while  the  control  mice 
died. 

The  possibility  of  conferring  immunity  by  means  of 
the  milk  of  an  immune  animal  is  further  shown  by  the 
experiments  of  Brieger  and  Ehrlich  (1892).  A  female 
goat  was  immunized  against  tetanus  by  the  daily  injec- 
tion of  "  thymus-tetanus  bouillon."  The  dose  was  grad- 
ually increased  from  0.2  c.c.  to  10  c.c.  At  the  end  of 
thirty-seven  days  a  mouse,  which  received  0.1  c.c.  of  the 
milk  of  this  goat  in  the  cavity  of  the  abdomen,  proved  to 
be  immune  against  tetanus.  Further  experiments  gave  a 
similar  result,  even  when  the  milk  of  the  goat  was  not 
injected  into  the  peritoneal  cavity  of  the  mouse  until  sev- 
eral hours  after  inoculation  with  a  virulent  culture  of  the 
tetanus  bacillus. 

When  the  casein  of  the  milk  was  separated  it  retained 


54  IMMUNITY   AND   SERUM-THERAPY. 

its  full  immunizing  activity,  and  by  concentration  in 
vacuo  a  thick  milk  was  obtained  which  had  a  very  high 
immunization  value — 0.2  c.c.  of  this  milk  protected  a 
mouse  against  forty-eight  times  the  lethal  dose  of  a  teta- 
nus culture. 

In  a  subsequent  communication  (1893)  Brieger  and 
Ehrlich  describe  their  method  of  obtaining  the  antitoxin 
of  tetanus  from  milk  in  a  more  concentrated  form.  Thej^ 
found  by  experiment  that  it  was  jDrecipitated  by  ammo- 
nium sulphate  and  magnesium  sulphate.  From  twenty 
seven  to  thirty  per  cent,  of  ammonium  sulphate  added  to 
milk  caused  a  precipitation  of  the  greater  part  of  the 
antitoxin.  This  precipitate  was  dissolved  in  water,  dia- 
lyzed  in  running  water,  then  filtered  and  evaporated  in 
shallow  dishes  at  35°  C.  in  a  vacuum.  One  litre  of  milk 
from  an  immune  goat  gave  about  1  gm.  of  a  transparent, 
yellowish  -  white  precipitate,  which  contained  fourteen 
per  cent,  of  ammonium  sulphate.  This  precipitate  had 
from  four  hundred  to  six  hundred  times  the  potency  of 
the  milk  from  which  it  was  obtained  in  neutralizing  the 
tetanus  toxine. 

In  a  still  later  communication  (1893)  Brieger  and  Cohn 
give  an  improved  method  of  separating  the  antitoxin 
from  the  precipitate  thrown  down  with  ammonium  sul- 
phate. The  finely  pulverized  precipitate  is  shaken  up 
with  pure  chloroform,  and  when  this  is  alloAved  to  stand 
the  antitoxin  rises  to  the  surface  while  the  ammonium 
salt  sinks  to  the  bottom.  By  filling  the  vessel  to  the 
margin  with  chloroform,  the  antitoxin  floating  on  the 
surface  can  be  skimmed  ofi",  after  which  it  qnickh'  dries. 
By  this  method  the  considerable  loss  which  occurred  in 
the  dialyzer,  used  in  the  previously  described  method,  is 
avoided. 


ACQUIRED   IMMUNITY.  55 

A  most  interesting-  question  presents  itself  in  connec- 
tion with  the  discovery  of  the  antitoxins.  Does  the  ani- 
mal which  is  immune  from  the  toxic  action  of  any  jDartic- 
ular  toxalbumin  also  have  an  immunity  for  other  toxic 
proteids  of  the  same  class  ?  The  experimental  evidence 
on  record  indicates  that  it  does  not.  In  Ehrlich's  exper- 
iments with  ricin  and  abriu  he  ascertained  that  an  animal 
which  had  been  made  immune  against  one  of  these  sub- 
stances was  quite  as  suscejitible  to  the  toxic  action  of  the 
other  as  if  it  did  not  possess  this  immunity,  i.e.,  the  anti- 
toxin of  ricin  does  not  destroy  abrin,  and  vice  versa.  As 
an  illustration  of  the  fact,  he  states  that  in  one  experi- 
ment a  rabbit  was  made  immune  for  ricin  to  such  an  ex- 
tent that  the  introduction  into  its  eye  of  this  substance 
in  powder  produced  no  inflammatory  reaction  ;  but  the 
subsequent  introduction  of  a  solution  of  abrin,  of  1  to 
10,000,  caused  a  violent  inflammation.  In  this  connection 
we  may  remark  that  there  is  some  evidence  to  show  that 
persons  w^ho  are  repeatedly  stung"  by  certain  poisonous 
insects — mosquitoes,  bees — acquire  a  greater  or  less  de-  > 
gree  of  immunity  from  the  distressing  local  effects  of 
their  stings. 

We  have  also  experimental  evidence  that  animals  may 
acquire  a  certain  degree  of  immunity  from  the  toxic 
action  of  the  venom  of  the  rattlesnake.  This  was  first 
demonstrated  by  Sewall  (1887),  and  has  been  recently 
confirmed  by  Calmette  (1894).  In  his  paper  detailing 
the  results  of  his  experiments  the  author  last  named 
says  : 

"  Animals  may  be  immunized  against  the  venom  of 
serpents  either  by  means  of  repeated  injections  of  doses 
at  first  feeble  and  progressively  stronger,  or  by  means  of 
successive  injections  of  venom  mixed  with  certain  chemi- 


66  IMMUNITY   AND   SERUM-THERAPY. 

cal  substances,  among-  which  I  mention  especially  chlo- 
ride of  gold  and  the  hypochlorites  of  lime  or  of  soda," 

"  The  serum  of  animals  thus  treated  is  at  the  same  time 
preventive,  antitoxic,  and  therai^eutic,  exactly  as  is  that 
of  animals  immunized  against  diphtheria  or  tetanus." 

"  If  we  inoculate  a  certain  number  of  rabbits,  under 
the  skin  of  the  thigh,  with  the  same  dose,  1  milligr. 
of  cobra  venom  for  example,  and  if  we  treat  all  of  these 
animals,  with  the  exception  of  some  for  control,  by  sub- 
cutaneous or  intraperitoneal  injections  of  the  serum 
of  rabbits  immunized  against  4  milligrs.  of  the  same 
venom,  all  of  the  control  animals  not  treated  will  die 
within  three  or  four  hours,  while  all  of  the  animals  will 
recover  which  receive  5  c.c.  of  the  therapeutic  serum 
within  an  hour  after  receiving-  the  venom." 

The  experimental  evidence  recorded  justifies  the  con- 
clusion that,  in  the  diseases  referred  to,  acquired  immu- 
nity depends,  chiefly  at  least,  upon  the  presence  of  a  pe- 
culiar proteid  substance  in  the  blood  of  the  immune 
animal — antitoxin — which  neutralizes  the  toxic  substance 
— toxin  or  toxalbumin — to  which  the  morbid  phenomena 
which  characterize  the  disease  are  due. 

But  it  would  be  premature  to  infer  that  in  all  infec- 
tious diseases  immunity  depends  upon  the  production  of 
an  antitoxin  in  the  blood  of  the  immune  animal.  Indeed 
we  have  experimental  evidence  which  shows  that  in  cer- 
tain cases  the  blood-serum  of  immune  animals  has  no 
antitoxic  power,  but  acts  upon  the  germ  itself,  instead  of 
upon  its  toxic  products. 

As  a  rule  the  antitoxins  have  no  bactericidal  action ; 
but  it  has  been  shown  by  the  experiments  of  Gamaleia, 
Pfeifi'er,  and  others,  that  in  animals  which  have  an  ac- 
quired immunity  against  the  spirillum  of  Asiatic  cholera 
and  against  spirillum  Metchnikovi,  there  is  a  decided  in- 


ACQUIRED   IMMUNITY.  57 

crease  in  tlie  bactericidal  power  of  the  blood-serum,  and 
that  immunity  probably  depends  upon  this  fact. 

The  researches  of  Metchnikoff  upon  hog  cholera,  of 
Issaeif  upon  pneumonia,  and  of  Sanarelli  upon  typhoid 
fever  indicate  that  the  immunity  conferred  upon  suscep- 
tible animals  by  ijrotective  inoculations  is  not  due  to  an 
antitoxin  but  to  a  substance  present  in  the  blood  of  im- 
mune individuals  which  acts  directly  upon  the  patho- 
genic microiJrg-anism,  as  is  the  case  in  cholera-immune 
animals.  The  animals  immunized  are  said  to  be  quite  as 
sensitive  to  the  action  of  the  bacterial  poisons  as  are 
those  which  have  not  received  protective  inoculations. 
"  Their  serum  does  not  protect  against  the  toxin,  but 
against  the  microbe  "  (Roux). 

Certain  important  questions  present  themselves  in 
connection  with  the  production  of  antitoxins  and  germi- 
cidal substances  in  the  blood  of  immune  animals,  one  of 
which  is :  Is  the  production  of  the  antitoxin  continuous 
while  immunity  lasts,  or  does  it  occur  only  during  the 
modified  attack  which  results  from  inoculation  with  an 
attenuated  virus,  or  of  filtered  cultures,  the  antitoxin 
being  subsequently  retained  in  the  circulating  blood  ? 
The  latter  supposition  does  not  appear  very  plausible, 
but  it  must  be  remembered  that  these  antitoxins  do  not 
dialyze  —  i.e.,  they  do  not  pass  through  animal  mem- 
branes— and  consequently  would  not  readily  escape  from 
the  blood-vessels,  notwithstanding  the  fact  that  thej^  are 
held  in  solution  in  the  circulating  fluid.  On  the  other 
hand,  the  passage  of  the  tetanus  antitoxin  into  the 
mother's  milk  would  indicate  a  continuous  supply,  other- 
wise the  immunity  of  the  mother  would  soon  be  lost. 
Further  experiments  are  required  to  settle  this  question 
in  a  definite  manner,  and  also  to   determine  the   exact 


58  IMMUNITY    AND   SERUM-THERAPY. 

source  of  the  antitoxins  in  the  animal  body  and  the 
modus  operandi  of  their  production.  According  to  Buch- 
ner  (1894)  the  antitoxins  are  not  to  be  regarded  as  reac- 
tive i^roducts  developed  in  the  body  of  the  immune  ani- 
mal, but  as  modified,  changed,  and  "  e)it<jiftete  "  products 
of  the  specific  bacterial  cells.  He  insists  that  they  do 
not  neutralize  the  toxins  by  direct  contact,  but  only 
through  the  medium  of  the  living  organism.  This  ex- 
planation scarcel}'  appears  tenable  in  view  of  the  experi- 
mental evidence,  and  the  fact  that  the  antitoxin  of  teta- 
nus escapes  in  considerable  quantity  with  the  milk  of  an 
immune  goat  without,  apparently,  diminishing  the  im- 
munity of  the  animal.  In  the  immunity  against  the  toxic 
action  of  the  vegetable  toxalbumins — ricin  and  abrin — as 
shown  by  Ehrlich's  experiments,  there  are  no  ' '  products 
of  bacterial  cells  "  introduced  with  the  pure  toxalbumin 
from  the  castor  bean  or  the  jequirity  bean  ;  and  we  have 
sufliciently  numerous  experiments  to  show  that  immu- 
nity, with  the  presence  of  antitoxins  in  the  blood,  may  be 
induced  by  precipitated  and  purified  toxalbumins  from 
filtered  cultures.  Several  of  the  experimenters,  also, 
have  reported  that  the  toxins  from  bacterial  cultures  are 
neutralized  in  vitro  hj  blood-serum  from  an  immune  ani- 
mal, or  by  the  precipitated  antitoxin  from  such  serum 
after  contact  for  a  certain  number  of  hours.  If  they  are 
correct  in  the  statement  that  a  certain  time  is  required 
after  the  antitoxin  has  been  brought  in  contact  with  the 
toxin,  in  order  that  the  latter  may  be  neutralized,  as 
shown  by  injection  of  the  mixture  into  a  suscej)tible  ani- 
mal, then  we  must  admit  that  this  neutralizing  effect  oc- 
curs outside  of  the  body  of  the  animal,  as  has  been  gen- 
erally assumed. 

The  experiments    of    Vaillard    are   also    opposed    to 


ACQUIEED   IMMUNITY.  59 

Bucliner's  view.  He  reports  that  in  a  rabbit  immunized 
against  tetanus,  "  a  volume  of  blood  equal  to  the  total 
amount  which  circulates  in  its  body  may  be  withdrawn 
without  diminishing,  in  an  appreciable  manner,  the  anti- 
toxic power  of  its  serum.  Therefore  the  antitoxin  must 
be  reproduced  as  fast  as  it  is  withdrawn."  The  author 
from  whom  we  have  just  quoted  (Roux)  also  reports  the 
results  of  experiments  which  show  that  the  antitoxic 
value  of  the  serum  of  a  rabbit  immunized  against  teta- 
nus does  not  bear  a  direct  relation  to  the  quantity  of  the 
tetanus  toxin  introduced,  but  depends  also  upon  the 
method  adopted.  When  a  few  large  doses  are  given  the 
result  is  far  less  favorable  than  that  obtained  by  giving 
the  same  amount  in  repeated  small  doses.  The  serum  of 
an  animal  immunized  by  thirty-three  small  doses  was 
found  to  neutralize,  in  vitro,  150  parts  of  toxin,  while  that 
of  an  animal  which  received  the  same  amount  in  nine 
doses,  only  neutralized  25  parts  of  the  same  toxin.  On 
the  other  hand  we  have  experiments  which  indicate  that 
the  supposed  neutralization  of  a  toxin  by  an  antitoxin,  in 
vitro,  is  not  really  a  chemical  neutralization.  Thus  Buch- 
ner  found  in  his  experiments  with  the  tetanus  toxin  and 
antitoxin,  in  a  dry  powder,  that  when  mixed  in  a  certain 
proportion  and  injected  into  white  mice  no  tetanic  symp- 
toms were  induced.  But  the  same  mixture  gave  rise  to 
distinct  tetanic  symptoms  in  guinea-pigs,  showing  that 
the  inference  that  the  toxin  had  been  neutralized  in  vitro, 
based  upon  the  experiment  on  mice,  woidd  have  been  a 
mistake.  And  certain  observations  made  by  Eoux  and 
Vaillard  seem  to  give  support  to  the  view  that  neutral- 
ization does  not  occur  in  vitro,  but  that  the  result  de- 
pends upon  some  physiological  reaction  induced  by  the 
antitoxin  within  the  body  of  the  living  animal.     These 


60  IMMUNITY   AND   SERUM-THERAPY. 

bacteriolog-ists  found  that  wlien  the  antitoxin  was  ap- 
parently in  excess,  tetanic  symptoms  could  be  induced 
in  susceptible  animals  if  they  had  been  in  any  way 
exhausted  prior  to  the  injection  of  the  mixture  of  toxin 
and  antitoxin;  and  that  the  same  result  followed  when 
their  resisting  power  had  been  reduced  by  injecting-  into 
them  at  the  same  time  filtered  cultures  of  other  bac- 
teria. 

In  this  connection  the  results  reported  by  Calmette, 
Phisalix,  and  Bertrand  are  of  interest.  These  investiga- 
tors found  that  when  the  antitoxin  of  snake-poison  was 
mixed  with  this  venom  in  a  proportion  which  neutralized 
its  toxic  j)roperties,  as  shown  by  experimental  inocula- 
tions, and  the  mixture  then  heated  to  70°  C,  by  which 
temperature  the  antitoxin  is  destroyed,  subsequent  inoc- 
ulations showed  that  the  toxin  was  still  active. 

The  experiments  of  Stern  (1894)  show  that  the  typhoid 
bacillus  not  only  grows  in  blood-serum  from  a  typhoid 
convalescent,  which  has  been  proved  to  neutralize  its 
pathogenic  effects  when  injected  into  a  susceijtible  ani- 
mal, but  also  that  its  toxic  products  are  developed  in  this 
culture  medium.  From  this  Stern  concludes  that  the 
serum  must  in  some  way  act  upon  the  infected  animal, 
causing  changes  which  enable  it  to  resist  infection,  rather 
than  upon  the  bacillus  or  upon  its  toxic  products  directly. 
It  has  also  been  shown  by  Behring  (1890)  for  the  diph- 
theria bacillus,  by  Vaillard  for  the  tetanus  bacillus  (1892), 
and  by  Issaeff  (1893)  for  the  micrococcus  of  pneumonia, 
that  these  several  pathogenic  microorganisms  may  be 
cultivated  in  the  blood-serum  of  animals  immunized  for 
the  diseases  which  they  produce. 

We  must  admit  that  the  exact  source  and  method  of 
production   of  the   antitoxins  in  the   animal  body,  and 


ACQUIRED   IMMUNITY.  61 

their  mode  of  action,  are  still  undetermined ;  and,  for  the 
present,  we  must  be  satisfied  with  the  knowledge  that  in 
some  way  these  so-called  antitoxins,  which  have  been 
proved  to  be  present  in  the  blood-serum  of  immune  ani- 
mals protect  these  animals  from  infection  by  pathogenic 
bacteria.  And  that  Avhen  transferred  to  susceptible  ani- 
mals they  confer  upon  them  a  temporarj^  immunity  ;  or  if 
introduced  after  infection  may  neutralize  the  pathogenic 
action  of  the  toxins  produced  by  specific  "disease 
germs." 

Finally,  there  is  experimental  evidence  to  show  that 
immunity  from  the  pathogenic  action  of  certain  bacteria 
may  be  produced  by  previous  injections  of  cultures  of 
other  bacteria  (sterilized  or  otherwise),  and  even  by  the 
injection  of  the  blood-serum  of  normal  individuals  or  of 
other  substances. 

Pasteur,  in  1880,  communicated  to  the  French  Acad- 
emy of  Sciences  the  results  of  experiments  which  led 
him  to  the  conclusion  that  fowls  which  had  an  acquired 
immunity  against  chicken  cholera  also  had  an  immunity 
against  anthrax.  And  Eoux  has  recently  reported  that 
the  blood-serum  of  a  horse  which  has  been  immunized 
against  tetanus  neutralizes  the  toxic  power  of  cobra 
poison.  But  the  contrary  effect  is  not  produced— i.e., 
the  blood-serum  of  an  animal  immunized  against  the 
cobra  poison  does  not  neutralize  the  tetanus  toxalbu- 
min.  The  statement  is  also  made  that  the  blood-serum 
of  a  rabbit  which  has  been  made  immune  against  hydro- 
phobia will  protect  a  susceptible  animal  against  the 
cobra  venom  in  doses  four  or  five  times  as  large  as  the 
usually  lethal  dose.  Also  that  rabbits  which  have  been 
immunized  against  snake-poison  are  less  susceptible  to 
the  toxic  effects  of  abrin  and  the  reverse — i.e.,  antiabrin 


62  IMMUNTTY    AND   SERUM-TIIEKAPY. 

neutralizes,  to  some  extent  at  least,  the  toxic  action  of 
snake-poison. 

The  writer  in  his  "  Report  on  the  Etiology  and  Preven- 
tion of  Yellow  Fever  "  (1890),  gives,  on  pp.  196  and  197, 
experimental  evidence  which  shows  that  the  injection 
into  the  peritoneal  cavity  of  rabbits  of  cultures  of  Bacil- 
lus pyocj'aneus  or  of  Bacillus  gracilis,  protected  the  ani- 
mals from  the  fatal  results  of  subsequent  injections  of  my 
bacillus  X,  which  was  extremely  fatal  to  rabbits  when  in- 
jected iijto  the  cavity  of  the  abdomen  in  doses  of  1  or  2 
c.c.  In  referring  to  these  experiments  I  say  :  "The  evi- 
dence favors  the  view  that  death  results  from  peritonitis 
(and  toxaemia?)  induced  by  iutra-peritoneal  injections, 
and  that  a  tolerance  on  the  part  of  the  peritoneum  may  he 
established  by  the  injection  of  certain  other  bacilli,  or  of 
sterilized  cultures  of  bacillus  X." 

This  corresponds  with  facts  subsequently  developed 
by  Issaeff  (1894)  in  his  experiments  with  reference  to  im- 
munity in  guinea-pigs  against  cholera  cultures  injected 
into  the  cavity  of  the  abdomen.  He  found  that  a  certain 
degree  of  immunity  was  established  by  the  previous  in- 
jection of  blood-serum  from  normal  individuals,  and  also 
of  various  acids,  alkalies,  and  neutral  liquids.  The  im- 
munity iDroduced  in  this  w^ay  was,  however,  feeble  and 
temporary,  and  could  not  properly  be  considered  as  iden- 
tical with  that  produced  by  inoculations  with  attenuated 
cultures  which  give  rise  to  a  mild  attack  of  a  specific  dis- 
ease. 

Referring  to  my  observation  that  injections  of  a  cult- 
ure of  Bacillus  pyocyaneus,  either  sterilized  or  otherwise, 
gave  rabbits  an  immunity  from  the  pathogenic  action  of 
my  bacillus  X,  I  may  call  attention  to  the  more  recent 
experiments  of  Rumpf  (1893)  and  of  Kraus  and  Buswell 


ACQUIRED   IMMUNITY.  63 

(1894),  with  reference  to  the  treatment  of  typhoid  fever 
with  sterilized  cultures  of  this  bacillus.  And  in  this  con- 
nection would  remark  that  my  bacillus  X  belongs  to  the 
same  group  as  the  typhoid  bacillus  and  Bacillus  coli  com- 
munis, but  is  more  decidedly  pathogenic  than  either  of 
these.  Cesaris-Demel  and  Orlaudi  have  recently  (1894) 
reported  their  success  in  immunizing-  animals  against 
infection  by  the  typhoid  bacillus  by  means  of  sterilized 
cultures  of  Bacillus  coli  communis,  and  the  reverse. 

While  this  chapter  relates  especially  to  acquired  im- 
munity from  infectious  diseases,  and  this  immunity  has 
been  shown  to  depend,  in  a  number  of  these  diseases 
at  least,  upon  the  development  of  antitoxins  in  the 
body  of  the  immune  animal,  it  may  be  worth  while  to 
refer  briefly,  before  closing,  to  some  examples  of  ac- 
quired immunity  of  a  different  order.  We  refer  to  the 
tolerance  of  extremes  of  heat  and  cold  which  may  be  es- 
tablished by  habitual  exposure,  and,  more  especially,  to 
the  tolerance  to  narcotics  and  irritant  poisons,  which  is 
very  remarkable  and  has  never  been  explained  in  a  satis- 
factory manner.  A  recent  writer  (Samuel,  1892)  has  pre- 
sented experimental  evidence  which  shows  that  the  local 
inflammation  which  results  from  the  application  of  cro- 
ton-oil  to  the  ear  of  a  rabbit  does  not  occur  when  a  sec- 
ond application  is  made  to  the  same  ear  after  recovery 
from  the  effects  of  the  first.  That  a  tolerance  may  be  ac- 
quired to  comparatively  large  doses  of  arsenic  is  well 
known,  and  the  tolerance  which  the  victims  of  drug 
habits  acquire  to  enormous  doses  of  narcotics  is  a  matter 
of  daily  observation.  In  the  writer's  paper  on  acquired 
immunity,  published  in  1881,  from  which  extended  quota- 
tions have  been  made  (i3p.  41-45),  an  attempt  is  made  to 
account  for  acquired  immunity  in  infectious  diseases  as 


64  IMMUNITY    AND   SERUM-THEllAPY. 

analogous  to  the  immnuity  to  drugs  just  referred  to  ; 
but  tlie  experimental  evidence  presented  in  the  present 
chapter  shows  that  the  analogy  has  no  scientific  founda- 
tion in  the  absence  of  any  evidence  that  there  is  an  an- 
titoxin of  morphia,  of  cocaine,  of  narcotin,  etc.,  in  the 
blood  of  the  hccbitiiis  of  these  drugs. 


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Ueber  die  bakteiuen-vernicbtenden  Eigenscbaften  des  Blutes  und 

ibre  Bezicbungen  zur  Immuuilat.     Centralbl.  ftir  Bakteriol.,  Bd. 

Y"I.,  1889. 
Ueber  die  Ursacben  der  Immunitat.     Fortscbr.  der  Med.,  Bd. 

VIII.,  1890,  No.  17. 
■  Ueber  Immunitat  und  Scbutzimpfung.    Tbiermed.  Yortrage  ber- 

ausgeg.  von  Dr.  Georg  Scbneidemubl,  1892,  Heft  11. 
Untersucbuuscen  iiber  die  Ursacben  der  ansreboreiien  und  erwor- 


benen   Immunitat.      Zeitscbr.    fiir   klin.   Med.,  1891.  (Sep.  Abdr. 

163  pp.) 
Maiselis,  J.  :  Ueber  die  erworbene  Immunitat  nacli  menscblichen  In. 

fektionskrankbeiten.     Inaugural  Dissertation.    8vo.  p.  29.     Berlin, 

1893. 
Massart,  J.  :  Le  cbimiotaxisme  des  leucocytes  et  I'immunite.    Annalcs 

de  rinst.  Pasteur,  1892,  p.  321. 
Massart  et  Bordet  :  Le  cbimiotaxisme  des  leucocytes  et  I'infection  mi- 

crobienne.     Annales  de  I'lnst.  Pasteur,  t.  v.,  1891.  p.  417. 
Masse :   Des  inoculations    preventives    dans    les    maladies    virulentes. 

Paris.  1883. 
^letschuikofE  :    Ueber  ein  Sprosspilzkrankbeit   der   Dapbnien.      Yir- 

cbow's  Arcbiv.,  Bd.  XCVI.,  p.  177. 
Sur  la  lutte   des  cellules   de   rorganisme   contre  I'invasion  des 

microbes.     Annales  de  I'Inst.  Pasteur,  t.  i.,  1887,  p.  821. 
Etudes  sur  I'immunite.     Annales  de  I'Inst.  Pasteur,  t.  iii.,  1889. 

p.  289  ;  ibid.,  t.  iv.,  p.  65  ;  ibid.,  t.  iv.,  p.  193  ;  ibid.,  t.  v.,  p.  465. 
Deux  travaux  du  laboratoire  de  M.  Baumgarten,  diriges  contre 

la  tbeorie  des  pbagocj'tes.     Ibid.,  t.  iv.,  1890,  p.  35. 
Recbercbes  sur  I'accoutumance  aux  produits  microbieus.     Ibid. . 


t.  v.,  1891.  p.  567. 


ACQUIRED   IMMUNITY.  69 

Metsclinikoff  :  L'immunite  dans  les  maladies  infectieuses.     La  Semaine 

Medicale,  1893,  p.  469. 
Ueber  Muskelphagocytose.     Centralbl.  f.  Baeteriol.,  XII.,  294. 

1892. 
Eludes   sur   rimmunite.     Immuiiite  des  lapins   vaccinee  contie 

le  microbe  du  hog-cholera.     Aimales  de  I'lnst.   Pasteur,  1892,  p. 

289. 

L'etat  actuel  de  la  question  de  rimmunite.     Auuales  de  I'lnst. 


Pasteur,  1894,  p.  706. 

Metschiukoff,  E.,  et  Soudakewitsch  :  La  phagocytose  musculaire.  An- 
nales  de  I'lnst.  Pasteur,  1892,  No.  1. 

Moore,  V.  A.  :  Can  the  Bacillus  of  Hog-cholera  be  Increased  in  Viru- 
lence by  Passing  it  through  a  Series  of  Rabbits  ?  Bureau  of  Ani- 
mal Industry,  Washington,  1894,  Bulletin  Xo.  6. 

Nissen  :  Zur  Kenntniss  der  bakterienvernichtenden  Eigenschaft  des 
Blutes.     Zeitschr.  fiir  Hygiene,  Bd.  VI.,  1889,  p.  487. 

Nuttall  :  Experimente  liber  die  bakterienfeindlichen  Einfliisse  des  thier- 
ischen  Korpers.     Zeitschr.  fiir  Hygiene,  Bd.  IV.,  1888,  p.  353. 

Ogata  -.  Ueber  die  bakterienfeindlichen  Substanz  des  Blutes.  Centralbl. 
fiir  Bakteriol.,  Bd.  IX.,  1891,  p.  597. 

Ogata  und  Jasuhara  :  Ueber  die  Einfliisse  einiger  Thierblutarten  auf 
Milzbrandbacillen.     Centralbl.  fiir  Baeteriol.,  Bd.  IX.,  p.  25. 

Pane,  N.  :  Ricerche  sulle  Sostanze  battericide  del  Siro  di  Sangue.  Ri- 
vista  Clin,  e  terapeutica,  1892,  No.  12. 

Pasteur  :  De  I'altenuation  du  virus  du  cholera  des  ponies.  Compte- 
rendu,  Acad,  des  Sci.,t.  xci.,  1880,  p.  673. 

De  I'attenuation  des  virus  et  de  leur  retour  a  la  virulence  ;  avec 

la  collaboration  de  MM.  Chamberland  et  Roux.  Ibid.,  xcii.,  1881, 
p.  429. 

Phisalix  et  Bertrand  .  Sur  la  propriete  antitoxique  du  sang  des  animaux 
vaccines  contre  le  venin  de  vipere.  Compte-rendu.  Acad,  des  Sci., 
^t.  cxviii.,  1894,  p.  356. 

Prudden  :  On  the  Germicidal  Action  of  Blood- serum  and  other  Body 
Fluids.     Medical  Record,  New  York,  January  25,  1890. 

Roux  :  Immunite  contre  la  septicemic  conferee  par  les  substances  solu- 
bles.    Annales  de  I'lnst.  Pasteur,  t.  i.,  1888,  p.  562. 

Immunite  contre   le   charbon  symptomatique  conferee   par  les 

substances  solubles.     Ibid.,  t.  ii. ,  p.  49. 

Sur  les  serums  antitoxiques.     Annales  de  I'lnst.  Pasteur,   1894, 


p.  722. 

Roux  et  Chamberland  :  Sur  l'immunite  contre  le  cliarbon  conferee  par 
les  substances  chimiques.     Ibid.,  t.  ii.,  p.  405. 

Salmon  and  Smith  :  Experiments  on  the  Production  of  Immunity  by 
Hypodermic  Injection  of  Sterilized  Cultures.  Centralbl.  fiir  Bak- 
teriol., Bd.  IL,  1887,  p.  543. 


70  IMMUNITY    AND   SEKUM-THEKAPY. 

Samuel,  S.  :  Ueber  c-ine  Art  vou  Immunitiit  nach  tlberstaudeuer  Croton- 

Enzfindung.     Yirchow's  Arcbiv,  CXXVIL,  Heft  3,  1893. 
Sanarelli :  Moyens  de  defense  de  I'organisme  coutre  les  microbes  apres 

vaccination  et  daus  la  guerisoa.     Auuales  de  I'lust.  Pasteur,  vii.. 

No.  3. 
Weitere  Mittbeilungen  iiber  Gifttbeorie  und  Pbagocytose.     Cen- 

tralbl.  fill-  Bakteriol.,  Bd.  X.,  1891,  p.  514. 
Sewell  :  Journal  of  Pbysiolog3S  vol.  viii.,  1887,  p.  203. 
.Sinjtiniu  :    Ueber   die   entwicklungsbeuimenden    Stoffwecbselprodukte 

der  Bakterien  und  die  sogenaunte  Reteutionsbypotbese.     Zeitscbr. 

fiir  Hygiene,  Bd.  IV.,  1888,  p.  202. 
Sniitb,  T.,  and  Moore,  V.  A.  :  Experiments  on  tbe  Production  of  Immu- 

iiitj^  etc.     Bureau  of  Animal  Industry,  Wasbington,  1894,  Bulletin 

No.  6. 
Stern  :  Ueber  die  Wirkung  des  menscblicben  Blutes  und  anderer  Korp- 

erflussigkeiten  auf  patbogenen  Bakterien.     Zeitscbr.  fiir  klin.  Med., 

Bd.  XVIII.,  1890. 
Sternberg  :  Explanation  of  Acquired  Immunity  in  Infectious  Diseases. 

American  Journal  of  tbe  Medical  Sciences,  April,  1881. 
Cbapter  on  "  Bacteria  in  Infectious  Diseases."    Bacteria,  Magnin 

and  Sternberg,  1884.  pp.  240-252. 
Manual  of  Bacteriology.    William  Wood  &  Co.,  New  York,  1892, 

pp.  225-262. 
Address  in  Medicine,  delivered  at  Yale  University,  June  23,  1893. 

Popular  Science  Montbly,  September,  1892. 
— • Practical  Results  of  Bacteriological  Researcbes.    Transactions  of 

tbe  Association  of  American  Pbysicians,  1893. 
Protective  Inoculations  in  Infectious  Diseases.     Transactions  of 


tbe  American  Public  Healtb  Association,  vol.  xviii.,  1893. 

Szekely,  A.  v.,  und  Szana,  A.  :  Experimentelle  Untersucbungen  iiber 
die  Veranderungen  der  sogenannten  mikrobiciden  Kraft  des  Blutes 
wabiend  und  nacli  der  Infection  des  Organismus.  Ceutralbl.  f. 
Bacterid.,  XII.,  Heft  3,  8,  4,  and  5,  1892.  * 

Vaugban  and  McClintock :  Tbe  Nature  of  tbe  Germicidal  Constituents 
of  Blood-serum.  Tbe  Medical  News,  Pbiladelpbia,  December, 
1893. 

Vaugban,  V.  C.  :  Tbe  Principles  of  Immunity  and  Cure  in  tbe  Infec- 
tious Diseases.     Medical  News,  1893,  vol.  ii.,  pp.  393,  421. 

Von  Fodor  :  Neuere  Untersucbungen  tiber  die  bakterientodtende  Wirk- 
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Bd.  VII.,  1890,  p.  753. 

Wagner,  K.  :  Zur  Lebre  von  der  Bedeutung  der  Temperature  bei  den 
Infektiou.skrankbeiten.  Wratscb.  1890,  No.s.  39-40  (Russian). 
Abst.  in  Ceutralbl.  fur  Bakteriol.,  Bd.  IX.,  p.  323. 


ACQUIRED   IMMUNITY.  71 

Wassermann,  A.  :    Ueber  Immunitiit  und  Giftfestigung.     Dtsche.  med. 

Wchschr.,  1892,  p.  369. 
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Archiv  fur  Anat.  und  Pliysiol.,  Bd.  III.,  1888,  p.  527. 
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rimmunita.     Giorn.  internaz.  delle  Sci.  med.,  1892,  p.  801. 


PART   SEOOI^D. 

PROTECTIVE  INOCULATIONS  IN  INFECTIOUS 
DISEASES  AND   SERUM-THEEAPY. 


I. 

ANTHRAX. 

The  discovery  of  the  anthrax  bacillus  by  Davaine 
(1863),  and  the  demonstration  of  its  etiological  relation  to 
the  disease  with  which  it  is  associated  by  the  researches 
of  Pasteur,  Toussaint,  Koch  (1878-1881),  and  other  pio- 
neers in  this  field  of  investig-atiou,  constitiite  the  founda- 
tion of  our  i^resent  knowledge  of  bacteriology  and  of  the 
practical  results  attained  in  protective  inoculations  and 
serum-therapy.  And  a  review  of  the  literature  relating 
to  the  anthrax  bacillus  would  show,  in  a  most  interesting- 
manner,  the  successive  steps  by  which  we  have  arrived  at 
the  important  results  which  have  gone  so  far  toward 
establishing  medicine  upon  a  scientific  basis.  In  the 
jjresent  volume,  however,  we  must  confine  our  attention 
to  those  investigations  which  relate  directly  to  the  sub- 
ject in  hand. 

Toussaint,  a  pioneer  in  researches  relating  to  protec- 
•  tive  inoculations,  has  a  short  paper  in  the  CompteH-Rendus 
of  the  French  Academy  of  Sciences  of  July  12,  1880,  en- 
titled "  Immunity  from  Anthrax  ( "  charbon  " )  Acquired 
as  a  Result  of  Protective  Inoculations." 

In  this  paper  he  announces  his  discovery  of  the  impor- 
tant fact  that  the  anthrax  bacillus  does  not  form  spores 
in  the  tissues  or  liquids  of  the  body  of  an  infected  ani- 
mal, but  multiplies  alone  by  binary  division — **  sa  multi- 
location  sefait  toujour s  par  une  divisio7i  du  myceliutny 


76  IMMUNITY    AND   SERUM-THERAPY. 

In  the  same  commuuication  he  reports  his  success  in 
conferring-  immunity  upon  five  sheep  by  means  of  protec- 
tive inoculations,  and  also  upon  four  j'ouug  clogs.  We 
must  therefore  accord  him  the  priority  in  the  publication 
of  experimental  data  demonstrating  the  practicability  of 
accomplishing  this  result. 

Toussaint  does  not  give  his  method  in  the  communica- 
tion above  referred  to,  but  the  following  quotation  from  a 
communication  made  to  the  Academy  of  Sciences  on 
March  19, 1881,  by  Pasteur,  shows  the  method,  and  at  the 
same  time  demonstrates  the  fact  that  Toussaint  was  the 
first  to  i^roduce  immunity  by  the  use  of  sterilized  cult- 
ures.    Pasteur  says  : 

"  By  inoculating  sheep  either  with  defibrinated  blood 
from  an  animal  dead  of  anthrax,  after  filtration  through 
several  thicknesses  of  paper,  or  with  the  same  blood  de- 
fibrinated and  subjected  to  55°  C.  for  ten  minutes,  accord- 
ing to  Toussaint,  these  sheep  subsequently  resist  inocula- 
tions with  anthrax  blood.  .  .  .  The  bacillus,  according 
to  Toussaint,  deposits  in  the  blood  of  animals  in  which  it 
multiplies  a  substance  which  may  become  its  own  vac- 
cine. By  filtration  w^hile  cold  in  one  case,  by  a  tempera- 
ture of  55°  C.  in  the  other,  the  bacillus  is  said  to  be  re- 
moved or  killed  ;  so  that  the  inoculation  of  filtered  or 
heated  blood  introduces  into  the  animal  inoculated  vac- 
cinal matter  depriv^  ed  of  bacteria." 

After  thus  stating  Toussaint's  method  and  ex]ilanation 
Pasteur  proceeds  to  raise  objections  against  this  method, 
the  principal  of  which  is  that  the  anthrax  bacillus  is  not 
killed  by  exj)OSure  to  a  temperature  of  55°  C.  for  ten  min- 
utes, and  that  inoculation  with  a  virus  prepared  in  this 
way  would  result  in  a  considerable  mortality  among  the 
animals  inoculated,  although  those  surviving  the  inocu- 
lation would  be  protected. 


ANTHRAX.  77 

In  a  communication  made  to  the  French  Academy  of 
Sciences,  September  27,  1880,  Pasteur  gave  an  account  of 
an  experiment  made  July  14,  1879,  upon  two  cows,  which 
in  connection  with  a  subsequent  experiment,  made  August 
6,  1880,  upon  four  cows,  led  him  to  the  conclusion  that  a 
single  attack  of  anthrax  protects  from  subsequent  attacks. 
He  says  in  the  paper  referred  to : 

"On  the  15th  of  September,  1880,  two  cows,  A  and  C, 
which  had  been  very  ill  as  a  result  of  the  first  inoculation, 
made  August  6th,  were  reinoculated  on  the  l^ft  side,  that 
is  to  say,  on  the  side  opposite  the  first  inoculation.  We 
used  five  drops  of  culture  of  the  bacillus  of  anthrax 
('  bacUridies  du  charbo?i ').  The  folloAving  days  there  was 
no  percei^tible  oedema  and  no  elevation  of  temperature 
in  either  cow.  The  question  is  then  resolved :  a  single 
attack  protects  ('  le  cliarhon  ne  recidive pas').'" 

The  next  important  steps  in  the  line  of  experimental 
research  leading  to  protective  inoculations  in  the  disease 
under  consideration- were  reported  by  Pasteur  in  his  com- 
munication to  the  French  Academy  made  at  the  seance  of 
February  28, 1881  (with  the  collaboration  of  Chamberland 
and  Eoux),  entitled  "  De  I'attenuation  des  virus  et  de 
leur  retour  a  la  virulence."  In  this  connection  Pasteur 
announces  his  discovery  of  the  fact  that  wdien  cultivated 
at  a  temperature  of  42°  to  43°  C,  the  anthrax  bacillus  no 
longer  forms  spores  and  rapidly  loses  its  virulence.  He 
says : 

"  As  regards  its  virulence,  the  extraordinary  fact  has 
been  ascertained  that  the  bacillus  is  no  longer  virulent 
after  it  has  been  kept  for  eight  days  at  a  temperature  of 
42°  to  43°  C.  ;  at  least  its  cultures  are  inoffensive  for  the 
guinea-pig,  the  rabbit,  and  the  sheep,  three  species  of  ani- 
mals which  are  very  susceptible  to  anthrax.     We  are 


*78  IMMUNITY    AND   SEHUM-THEIIAPY. 

able,  then,  not  only  to  attenuate  virulence  but  to  effect  its 
complete  extinction,  by  a  simple  method  of  cultivation, 

"  Before  the  extinction  of  its  virulence  the  microbe  of 
charbon  passes  through  the  intermediate  degrees  of  at- 
tenuation, and,  on  the  other  hand,  as  happens  also  with 
the  microbe  of  fowl  cholera,  each  of  these  degrees  of  vir- 
ulence may  be  reproduced  by  cultivation.  Finally,  as 
sliown  in  one  of  our  recent  communications,  since  one 
attack  of  anthrax  i^rotects,  each  one  of  our  attenuated  mi- 
crobes of  charbon  constitutes  a  vaccine  for  the  microbe 
of  superior  virulence  ;  that  is  to  say,  a  virus  suitable  to 
produce  a  mqre  benign  malady.  What,  then,  is  more  easy 
than  to  find  among  these  a  virus  suitable  to  give  anthrax 
to  sheep,  cows,  or  horses,  without  causing  them  to  perish, 
and  capable  of  preserving  them  from  a  subsequent  fatal 
attack  ?  We  have  already  practised  this  operation  upon 
sheep  with  great  success." 

At  the  end  of  this  important  communication  Pasteur 

says  : 

"  I  concluded  my  communication  of  October  26th  b\'  re- 
marking that  the  attenuation  of  virus  by  the  influence  of 
the  air  is  probably  one  of  the  factors  in  the  extinction  of 
great  epidemics.  The  facts  just  recorded,  in  their  turn, 
may  serve  to  explain  the  so-called  spontaneous  appear- 
ance of  these  scourges.  An  epidemic  which  has  been 
terminated  by  the  attenuation  of  its  virus  may  be  re- 
lighted by  the  reinforcement  of  this  virus  under  certain 
influences.  The  accounts  which  I  have  read  of  the  spon- 
taneous appearance  of  the  plague  appear  to  me  to  offer 
examples  of  this.  The  plague  is  a  virulent  malady  which 
prevails  in  certain  countries.  In  all  of  these  countries  its 
attenuated  virus  probably  exists,  ready  to  take  its  active 
form  when  the  necessary  conditions  as  to  climate,  famine, 
and  distress  again  prevail.  There  are  other  virulent  mal- 
adies which  appear  spontaneously  in  all  countries,  such 
as  camp  typhus.  Without  doubt  the  germs  of  the  mi- 
crobes which  cause  these  diseases  are  everywhere  distrib- 


ANTHRAX.  79 

uted.  Man  carries  them  about  him,  or  in  his  intestine, 
without  great  damage,  but  ready,  nevertheless,  to  become 
dangerous  when,  as  a  result  of  certain  conditions  or  of 
successive  development  upon  the  surface  of  wounds,  in 
bodies  enfeebled  or  otherwise,  their  virulence  is  progres- 
sively reinforced.  And  from  this  point  of  view  virulence 
appears  to  us  under  a  new  light  which  is  somewhat  dis- 
quieting for  humanity,  unless  nature,  in  the  evolution 
which  has  occurred  during  the  past  centuries,  has  already 
encountered  all  possible  occasions  for  the  production  of 
virulent  or  contagious  diseases,  an  assumption  which 
seems  very  improbable. 

"  What  is  an  inoffensive  microscopic  organism  for  man 
or  for  a  given  animal  1  It  is  an  organism  which  cannot 
develop  in  our  body  or  in  that  of  the  animal ;  but 
nothing  proves  that  if  this  microscopic  organism  should 
penetrate  into  some  other  of  the  thousands  of  species  of 
the  creation  it  could  not  invade  it  and  cause  it  to  become 
sick.  Its  virulence,  then,  reinforced  by  passing  through 
a  series  of  individuals  of  this  species,  might  become  such 
that  it  could  invade  man  or  one  of  the  domestic  animals. 
By  this  means  new  contagious  may  be  created.  I  am 
disposed  to  believe  that  it  is  in  this  way  that,  in  the 
course  of  ages,  have  appeared  small-pox,  syphilis,  the 
plague,  yellow  fever,  etc." 

This  broad  induction  has  received  considerable  sup- 
port from  more  recent  researches,  which  show  that  the 
typhoid  bacillus,  the  cholera  spirillum,  and  other  impor- 
tant pathogenic  bacteria  become  attenuated  when  they 
lead  a  saprophytic  existence  for  some  time,  and  regain 
their  virulence  when  they  are  j)ropagated  within  the 
bodies  of  susceptible  animals. 

In  a  later  communication  (March  21,  1881)  Pasteur 
says  that  he  .has  found  by  experiment  that  when  attenu- 
ated varieties  of  the  anthrax  bacillus  form  spores,  these 
again  reproduce  the  same   pathogenic  variety,  so  that 


80  IMMUNITY   AND   SEUUM-TJIEKAPY. 

cultures  of  each  degree  of  attenuation  can  be  maintained 
indefinitel3\ 

On  June  13,  1881,  Pasteur  communicated  the  results  of 
liis  famous  experiment  at  Pouilly-le-Fort,  near  Melun. 
He  says: 

"  On  the  5th  of  May,  1881,  we  inoculated,  by  means  of 
a  Pravaz  syring-e,  twenty-four  sheep,  one  goat,  and  six 
cows,  each'  animal  wdth  five  drops  of  an  attenuated  cult- 
ure of  the  anthrax  bacillus.  On  the  17th  of  May  we  re- 
inoculated  these  animals  with  a  second  virus,  also  atten- 
uated, but  more  A^irulent  than  the  first. 

"  On  the  31st  of  May  we  proceeded  to  make  a  very 
virulent  inoculation  in  order  to  test  the  efficacy  of  the 
preventive  inoculations  made  on  the  5th  and  7th  of  May. 
For  this  experiment  we  inoculated  the  thirty  vaccinated 
animals,  and  also  twenty-four  sheep,  one  goat,  and  four 
cows,  which  had  not  received  any  previous  treatment. 

"  The  verj^  virulent  virus  used  on  the  3lst  of  May  was 
obtained  from  spores  preserved  in  my  laboratory  since 
the  21st  of  March,  1877. 

"In  order  to  make  the  experiments  more  comparable 
we  inoculated  alternately  a  vaccinated  and  a  non-vacci- 
nated animal.  When  the  operation  was  finished  all  of 
those  present  were  invited  to  reassemble  on  June  2d,  i.e., 
forty-eight  hours  after  the  virulent  inoculation  was  made. 

"  Upon  the  arrival  of  the  visitors  on  June  2d,  all  were 
astonished  at  the  result.  The  twenty-four  sheep,  the 
goat,  and  the  six  cows  which  had  received  the  attenuated 
virus,  all  presented  the  appearance  of  health.  On  the 
contrary,  twenty  of  the  sheep,  and  the  goat,  which  had 
not  been  vaccinated,  were  already  dead  of  anthrax  ;  two 
more  of  the  non-vaccinated  sheep  died  before  the  eyes  of 
the  spectators,  and  the  last  of  the  series,  expired  before 
the  end  of  the  day.  The  non-vaccinated  cows  were  not 
dead.  AVe  had  previously  proved  that  cows  are  less 
subject  than  sheep  to  die  of  anthrax.  But  all  had  an  ex- 
tensive oedema  at  the  point   of  inoculation,  behind   the 


A  NTH  n  AX.  81 

shoulder.  Certain  of  these  oedematous  swellings  increased 
during-  the  following  days  to  such  dimensions  that  they 
contained  several  litres  of  liquid,  deforming  the  animal. 
One  of  them  even  nearly  touched  the  earth.  The  tem- 
perature of  these  cows  was  elevated  3°  C.  The  vaccinated 
cows  did  not  experience  any  elevation  of  temperature,  or 
tumefaction,  or  the  slightest  loss  of  appetite.  The  suc- 
cess, therefore,  was  as  complete  for  the  cows  as  for  the 
sheep." 

The  fact  that  infection  depends  to  some  extent  upon 
the  number  of  bacilli  introduced,  and  that  animals  which 
have  a  certain  degree  of  immunity,  like  the  Algerian  race 
of  sheep,  may  succumb  when  they  are  inoculated  with  a 
certain  quantity  of  virus,  although  they  resist  a  smaller 
amount,  was  announced  by  Chauveau  in  his  communica- 
tion to  the  French  Academy  at  the  seance  of  June  28, 
1880.     He  says : 

"  The  facts  which  I  have  just  presented  show  that  the 
anthrax  bacillus  behaves  in  the  organism  of  Algerian 
sheep  not  as  if  it  were  deprived  of  the  principles  neces- 
sary for  its  development,  but  rather  as  if  it  were  in  a 
medium  rendered  unsuitable  for  its  groAvth  by  the  pres- 
ence of  substances  injurious  to  it.  In  a  very  small 
number  the  bacilli  are  arrested  in  their  development  by 
the  inhibitory  influence  of  these  substances.  When  they 
are  very  numerous,  on  the  contrary,  they  surmount  more 
easily  this  obstacle  to  their  proliferation." 

This  quotation  shows  that  Chauveau  had  at  this  early 
date  arrived  at  an  explanation  of  immunity  very  nearly  in 
accord  with  that  which  is  now  generally  accei)ted. 

The  fact  that  infection  is  influenced  by  the  quantity  of 
the  infectious  material  introduced  had  previously  been  in- 
sisted upon  by  Davaine  in  his  paper  entitled  "  Recherches 
siir  qitelques  unes  des  conditions  qui  favorisent  ou  qui  em- 
6 


82  IMMUNITY   AND   SERU3I-THERAPY. 

pechoit  le  developemeni  de  la  septicemie"  published  in  tlie 
bulletin  of  the  Academy  of  3Iedicine,  seance  of  February 
18,  1879. 
Da  vain  e  says : 

"A  third  condition  relates  to  the  quantity  of  bac- 
teria introduced  into  the  tissues.  This  question  of  quan- 
tity has  been  made  manifest  in  our  experiments.  Not 
only  does  it  ditier  in  different  species  of  animals,  the 
rabbit  and  the  dog-,  for  example,  but  it  varies  in  different 
animals  of  the  same  species." 

In  his  communication  to  the  Academy  of  Sciences, 
made  on  April  4,  1881,  Chauveau  gives  the  results  of  his 
exi^eriments  in  j)roducing  immunity  by  inoculations  with 
very  small  quantities  of  virus.  After  some  preliminary 
experiments  with  a  larger  number,  five  sheep  were  inocu- 
lated with  diluted  anthrax  blood  estimated  to  contain 
two  hundred  and  fifty  bacilli  for  each.  All  of  the  ani- 
mals survived  the  inoculation  after  having  manifested 
some  slight  febrile  reaction.  Six  weeks  later  all  were 
reinoculated  with  a  dose  which  should  have  been  fatal 
to  an  unprotected  animal.  One  of  the  animals  died  of 
anthrax,  the  other  four  resisted  perfecth'. 

On  June  26,  1882,  Chauveau  reported  to  the  Academy 
of  Sciences  the  results  of  his  experiments  relating  to  the 
protection  of  animals  from  anthrax  infection  by  the 
method  of  Toussaint.  By  carefully  conducted  experi- 
ments Chauveau  found  that  nine  or  ten  minutes'  exposure 
to  a  temperature  of  54°  C.  killed  all  of  the  bacilli  in 
anthrax  blood,  and  the  same  result  was  obtained  by  six- 
teen minutes'  exposure  to  52°  C,  while  at  50°  C.  the  time 
required  is  twenty  minutes.  An  attenuated  virus  suitable 
for  protective  inoculations  is  obtained  by  exposure  for  a 
somewhat  shorter  time,  and  as  a  result  of  his  experi- 


ANTHRAX.  83 

ments  Chauveaii  was  led  to  the  conclusion  tliat  for  a  first 
inoculation  anthrax  blood  heated  to  50°  C.  for  fifteen 
minutes  afibrded  a  g-ood  attenuated  virus.  This  was  to 
be  followed  after  an  interval  of  ten  to  fifteen  days  by  a 
second  inoculation  with  a  stronger  virus,  obtained  by  ex- 
posing anthrax  blood  to  the  same  temperature  (50°  C.) 
for  nine  or  ten  minutes.  These  inoculations  sufficed  to 
protect  the  animals  when  they  were  subsequently  inocu- 
lated with  virus  of  full  strength — blood  from  an  animal 
which  had  recently  succumbed  to  the  disease.  Chauveau 
says  with  reference  to  this  method : 

"  In  one  hour,  with  a  single  guinea-pig  [dead  of  an- 
thrax], it  is  easy  to  prepare  the  quantity  of  vaccine  re- 
quired to  inoculate  more  than  five  hundred  sheep.  The 
inoculation  is  made  with  the  point  of  a  lancet,  charged, 
by  the  method  in  use  in  my  laboratory,  with  a  very  small 
quantity  of  virus.  Two  or  three  large  punctures  under 
the  skin,  upon  the  internal  surface  of  the  ear,  suffice  for  a 
successful  inoculation. 

"  The  vaccine  prepared  in  this  way  should  be  used  at 
once,  or  at  least  not  later  than  the  day  after  it  has  been 
prepared.  Experience  has  shown  me  that  it  is  then  quite 
as  harmless  and  quite  as  efficacious  as  Pasteur's  vaccine." 

In  the  preparation  of  an  attenuated  virus  by  this 
method  Chaiiveau  insists  upon  attention  to  the  follow- 
ing points  : 

"  The  first  rule  to  follow,  and  the  principal  one,  is  to 
practise  the  heating  in  such  a  manner  that  all  parts  of 
the  anthrax  blood  are  raised  to  the  required  temperature 
and  withdrawn  from  it  at  the  same  instant.  When  the 
quantity  of  blood  to  be  transformed  to  a  vaccine  is  too 
great,  all  parts  are  not  uniformly  acted  upon  by  the  very 
short  exposure  to  heat ;  the  virulent  agents  in  the  deeper 
layers  may,  in  that  case,  preserve  all  of  their  activity, 


84  IMMUNITY    AND   SERUM- THERAPY. 

and  cause  a  fatal  infection.  To  avoid  tins  it  is  best  to 
enclose  the  blood  in  little  cylindrical  pipettes,  1  mm.  in 
diameter.  The  extremity  of  these  pipettes  is  sealed, 
and  the  portion  which  contains  tfie  blood  is  immersed 
in  a  considerable  quantity  of  water  maintained  at  the 
proper  temperature.  At  the  end  of  the  proper  time  they 
are  taken  from  the  hot  bath  and  plunged  into  cold  water. 
"Another  rule  should  be  rigorously  observed  if  one 
wishes  to  be  sure  of  success.  The  blood  should  be  col- 
lected under  conditions  which  make  it  sure  that  the  viru- 
lent agents  introduced  into  the  tu])es  all  have  the  same 
vitality,  the  same  activit\%  and  that  they  are  impressed  in 
the  same  degree  by  the  heating.  This  is  the  case  when 
M'e  take  the  blood  from  a  guinea-pig  just  dead,  after  hav- 
«ing  survived  from  thirty-six  to  forty-eight  hours  an  in- 
oculation with  very  active  virus.  Before  introducing  the 
blood  into  the  pipettes  it  should  be  allowed  to  coagulates 
and  the  coagula  should  be  broken  and  crushed  in  order 
to  obtain  a  defibrinated  blood,  which  is  always  very  rich 
in  virulent  bacilli." 

In  a  subsequent  communication  (February  26,  1883), 
Chauveau  admits  that  the  application  of  this  method  is 
somewhat  difficult  and  delicate  when  blood  is  employed, 
and  states  that  it  is  far  more  satisfactory  to  use  pure  cult- 
ures, which  may  be  attenuated  in  the  same  way.  He  pre- 
fers to  cultivate  the  bacillus  in  a  bouillon  made  from  the 
flesh  of  a  chicken,  and  to  start  his  culture  by  adding  to 
this  bouillon  a  droj^  of  blood  from  an  animal  just  dead 
from  anthrax.  The  culture  is  left  for  twenty  hours  in  an 
incubating  oven  at  a  temperature  of  43°  C.  During  this 
time  there  is  an  abundant  development  of  the  bacillus, 
and  the  culture  is  ready  to  be  subjected  to  the  attenuat- 
ing action  of  a  higher  temperature.  This  is  accomplished 
by  exposure  to  a  temperature  of  47°  C.  for  a  period  of 
one,  two,  three,  or  four  hours,  according  to  the  degree  of 


ANTHRAX.  85 

attenuation  desired.  After  three  hours'  exposure  the  at- 
tenuated culture  no  longer  kills  guinea-pigs.  In  a  later 
communication  (March  5,  1883)  Chauveau  states  that  he 
has  ascertained  by  experiment  that  the  degree  of  attenu- 
ation produced  by  this  method  is  maintained  in  subse- 
quent cultures  made  at  43"  C,  from  the  attenuated  culture 
thus  obtained. 

Another  method  of  attenuating  the  virulence  of  anthrax 
cultures  is  that  described  by  Chauveau,  in  1885.  This 
consists  in  cultivating  the  bacillus  at  a  temperature  of 
88°  to  39°  C,  under  a  pressure  of  eight  atmospheres. 
Cultures  treated  in  this  way  killed  guinea-pigs,  but  did 
not  kill  sheep,  cattle,  or  horses,  and  constituted  a  suitable 
attenuated  virus  for  protective  inoculations  in  these  ani- 
mals. One  drop  was  used  for  a  sheep,  and  two  drops  for 
a  cow  or  a  horse,  and  the  immunity  was  proved  to  last 
for  a  year. 

Kitt,  in  experiments  made  in  1884  and  1885,  found  that 
an  attenuation  of  the  virulence  of  anthrax  bacilli  may  be 
effected  by  passing  them  through  birds,  which  have  but 
little  susceptibility  to  anthrax  infection  ;  but  the  results 
obtained  were  not  uniform,  and  the  method  was  not 
thought  to  have  any  great  practical  value.  In  the  same 
paper  Kitt  gives  an  account  of  his  experiments  with  Pas- 
teur's vaccine,  No.  1  and  No.  2,  which  he  obtained  from 
the  agent  in  Paris.  These  experiments  led  him  to  the 
conclusion  that  the  attenuated  cultures  used  by  Pasteur 
are  too  weak.  But  by  passing  them  through  guinea-pigs 
their  virulence  was  increased  so  that  they  served  to  pro- 
tect cattle  and  sheep,  although  not  without  danger  for 
the  last-mentioned  animals. 

During  the  year  1882  Pasteur's  method  was  extensive- 
ly iiractised  in   the   department  of  Eure-et-Loir,  where 


86  IMMUNITY   AND   SERUM-THERAPY. 

* 

anthrax  Avas  very  prevalent  r.:i(l  had  been  the  cause  of 
extensive  losses.  The  results  of  these  protective  inocu- 
lations were  reported  to  the  Academy  of  Sciences  (se- 
ance of  December  18,  1882)  by  Pasteur,  who  submitted, 
with  some  remarks,  a  report  prepared  by  M.  Boutet, 
from  which  we  quote  as  follows : 

"  The  number  of  sheep  vaccinated  during-  the  year  has 
been  7i),392  ;  among-  these  Hocks  the  average  annual  loss 
during  the  past  ten  years  was  7,237 — 9.01  per  cent.  Since 
the  vaccinations  but  518  animals  have  died — 0.65  per  cent. 
We  must  observe  that  this  year,  probably  on  account  of 
the  great  humidity,  the  mortality  in  Eure-et-Loir  has  only 
been  three  per  cent.  The  losses  should  therefore  have 
been  2,382,  instead  of  518,  without  the  vaccinations.  In 
the  flocks  which  were  only  partly  vaccinated  we  had  2,308 
vaccinated,  and  1,659  not  vaccinated  ;  the  loss  among  the 
first  was  8,  or  0.4  per  cent. ;  among  the  second  the  loss 
was  60,  or  3.9  per  cent.  We  call  attention  to  the  fact  that 
in  these  flocks,  in  different  cantons  of  the  department,  the 
sheep  vaccinated  and  not  vaccinated  were  subjected  to 
the  same  conditions  of  soil,  of  lodging,  of  food,  of  tem- 
perature, and  that  consequently  they  were  exposed  to 
identical  influences. 

"  The  veterinary  surgeons  in  Eure-et-Loir  ha-ve  vacci- 
nated 4,562  animals  of  the  bovine  species.  Out  of  this 
number  the  annual  loss  had  been  322.  Since  the  vaccina- 
tions only  11  cows  have  died.  That  is,  the  annual  mortal- 
ity has  been  reduced  from  7.03  per  cent,  to  0.24  per  cent. 

"  Some  engorgements,  generally  not  serious,  having  oc- 
curred after  vaccinating  horses,  and  the  mortality  not  be- 
ing great  in  this  species,  the  veterinarians  have  thought 
it  prudent  not  to  vaccinate  horses,  on  a  large  scale.  Only 
524  were  vaccinated  ;  three  of  these  died  after  the  first 
vaccination." 

Notwithstanding  this  favorable  report  some  bacteriol- 
ogists, and  notably  Koch,  were  not  disposed  to  admit 


ANTHRAX.  87 

the  practical  value  of  Pasteur's  anthrax  inoculations.  At 
the  conclusion  of  an  elaborate  memoir  published  in  the 
second  volume  of  "  Mittheilungen  "  of  the  Imperial 
Board  of  Health  of  Germany  (1884),  Koch  and  his  collab- 
orators (Gaffky  and  Loeffler)  say  : 

"  As  now  a  certain  immunity  ag-ainst  inoculated  anthrax 
cannot  be  obtained  by  the  method  of  Pasteur,  as  we  have 
seen,  without  considerable  losses,  and  as  the  immunity 
secured  at  the  expense  of  considerable  loss  is  only  an  im- 
perfect protection  against  contracting-  anthrax  in  the 
ordinary  way,  we  must  consider  the  protective  inocula- 
tions heretofore  practised  as  of  doubtful  utilit}^,  espe- 
cially when  we  remember  that  the  second  inoculation 
with  a  yet  stronger  virus  causes  the  death  of  more  ani- 
mals which  may  serve  to  further  spread  the  disease." 

The  attenuating  influence  of  lig-lit  on  the  anthrax  ba- 
cillus and  the  fact  that  cultures  attenuated  in  this  way 
may  be  used  for  protective  inoculations  was  first  ascer- 
tained by  Arloing  (1886).  Koux  subsequently  (1887) 
showed  that  the  presence  of  oxygen  is  a  necessary  factor 
in  the  sterilization  of  cultures  by  exposure  to  sunlight. 
Behring,  who  has  since  been  so  active  in  the  field  of  re- 
search to  which  the  present  volume  relates,  published  an 
article  in  the  Centralhlatt  fiXr  Tdinische  3Iedicm  in  1888 
(September  22d)  in  which  he  attempted  to  explain  the 
natural  immunity  of  white  rats  against  anthrax  infection. 
His  conclusions  are  given  as  follows : 

"  1.  The  blood-serum  of  white  rats  is  not  a  favorable 
medium  for  the  anthrax  bacillus." 

"  2.  The  blood-serum  of  rats  differs  from  that  of  ani- 
mals susceptible  to  infection  by  its  greater  alkalinity." 

"  3.  By  the  addition  of  an  acid  to  the  blood-serum  of 
rats  this  becomes  a  favorable  medium  for  the  growth  of 
the  anthrax  bacillus." 


88  IMMUNITY   AND    SERUM-TIIEKAPY. 

"  4.  The  blood-serum  of  rats  wliicli  are  treated,  during 
life,  hi  such  a  Avay  as  to  reduce  the  alkalinity  of  the 
blood  becomes  a  suitable  medium  for  the  development  of 
the  anthrax  bacillus." 

As  we  have  pointed  out  in  the  chapter  on  Natural  Im- 
munity (p.  28),  the  true  explanation  of  the  facts  ascer- 
tained in  Behring's  experiments  is  probably  to  be  found, 
not  in  the  germicidal  power  of  the  comparatively  small 
amount  of  alkali  present  in  the  rat's  serum,  but  in  the 
fact  that  the  germicidal  proteid  produced  by  the  leuco- 
cytes is  only  soluble  in  an  alkaline  medium.  In  a  paper 
published  in  the  Annales  of  the  Pasteur  Institute  (August, 
1888),  Roux  and  Chamberland  have  given  an  account  of 
experiments  made  by  them  which  establish  the  fact  that 
immunity  against  anthrax  may  be  established  by  inocu- 
lating susceptible  animals  with  blood  from  an  animal 
dead  from  anthrax,  in  which  the  anthrax  bacilli  had  been 
killed  by  heat  or  removed  by  filtration  (Sur  I'immunite 
contre  le  charbon  conferee  par  des  substances  chi- 
miques).  These  experiments  were  commenced  in  1881. 
The  authors  named  say  : 

"  In  repeating  the  experiments  of  Toussaint  upon  an- 
thrax blood  which  had  been  heated,  we  made  several  ob- 
servations which  convinced  us  that  it  is  possible  to  con- 
fer immunity  against  anthrax  upon  sheep  by  injecting 
under  their  skin  anthrax  blood  which  does  not  contain 
any  living  bacilli." 

While  immunity  was  produced  in  this  way,  Roux  and 
Chamberland  remark  that  the  sheep  which  had  received  a 
comparatively  large  dose  were  quite  sick  when  subse- 
quently inoculated  with  a  virulent  culture,  and  the  im- 
munity acquired  was  less  reliable  than  that  obtained  by 


ANTHRAX.  89 

Pasteur's  metliocl  with  two  vaccines  of  different  degrees 
of  attenuation. 

In  an  investigation  made  by  Hankin,  in  tlie  laboratory 
of  Professor  Koch  at  the  Hygienic  Institute  of  Berlin, 
the  results  of  which  are  given  in  a  preliminary  account 
published  in  the  British  Medical  Journal  (October  12, 
1889),  the  important  fact  was  ascertained  that  immunity 
may  be  produced  in  susceptible  animals  by  inoculating 
them  with  an  "albumose"  isolated  from  anthrax  cultures. 
Hankin  gives  the  following  account  of  his  method  of  ob- 
taining this  immunizing  proteid  from  anthrax  cultures  : 

"  In  the  course  of  my  process  of  preparation  it  is  pre- 
cipitated from  its  solution  by  the  addition  of  a  large  bulk 
of  absolute  alcohol,  and  well  washed  in  this  liquid  to  free 
it  from  ptomaines ;  it  is  well  known  that  all  such  sub- 
stances are  soluble  in  alcohol.  It  is  then  filtered  off  and 
dried  ;  then  it  is  redissolved  and  filtered  through  a 
Chamberland  filter.  A  rough  estimate  of  the  percent- 
age of  albumose  present  in  the  clear  solution  thus  ob- 
tained is  made  colorimetrically  by  means  of  the  biuret  re- 
action and  a  peptone  solution  of  known  strength." 

"In  one  experiment  four  rabbits  (Nos.  23  to  26)  were 
inoculated  subcutaneously  with  virulent  anthrax  spores. 
No.  26  served  as  a  control  and  died  in  about  forty  liours. 
The  other  three  rabbits  had  the  albumose  solution  in- 
jected into  the  ear-vein  at  the  same  time.  Nos.  24  and 
25  each  had  about  the  five  -  millionth  of  their  body- 
weight,  while  No.  23  had  only  the  ten-millionth  of  its 
body-weight  of  albumose.  No.  25  died  in  less  than  forty- 
eight  hours,  but  Nos.  23  and  24  survived.  Ten  days  later 
Professor  Koch  kindly  reinoculated  these  two  rabbits  for 
me  with  very  virulent  anthrax  from  an  agar-agar  culture. 
Their  temperature  has  remained  normal  since  then,  and 
they  are  now  alive  and  well  a  fortnight  after  this  opera- 
tion. I  have  also  succeeded  in  producing  immunity  in 
mice  against  attenuated  anthrax." 


90  IMMUNITY   AXD   SEKUM-TIIERAPY. 

In  a  paper  published  in  the  Proceedings  of  the  Royal 
Society  in  1890,  Dr.  Sidney  Martin  has  given  an  account 
of  his  researches  relating-  to  "  The  Chemical  Product*  of 
the  Growth  of  Bacilhis  Anthracis,  and  their  Physiologi- 
cal Action."  In  his  experiments  the  cultures  were  main- 
tained for  from  ten  to  fifteen  days,  and  the  bacilli  were 
then  removed  by  filtering  through  a  Chamberland  filter. 
The  filtrate  was  found  to  contain  : 

''1.  Proto-albumose,  deutero-albumose,  and  a  trace  of 
peptone,  all  with  the  same  chemical  reactions  as  the  sim- 
ilar bodies  formed  in  peptic  digestion.  2.  An  alkaloid. 
3.  Small  quantities  of  leucin  or  tyrosin.  The  chief  char- 
acteristic of  the  proto-  and  deutero-albumose  obtained 
from  anthrax  cultures  was  found  to  be  their  strong  alka- 
linity in  solution.  This  was  not  removed  by  prolonged 
dialj'sis  or  by  washing  in  alcohol,  chloroform,  benzene 
or  ether.  These  proteids  are  precipitated  in  an  alkaline 
condition  by  saturation  with  NaCl  (proto  -  albumose)  or 
(NHJ2S0,." 

The  alkaloid  found  was  soluble  in  water  or  in  absolute 
alcohol,  was  strongly  alkaline  in  solution,  and  readily 
formed  salts  with  acids.  It  was  slightly  volatile  and  lost 
its  poisonous  properties  to  a  great  extent  when  exposed 
to  the  air  for  some  time.  A  mixture  of  the  two  albumoses 
was  toxic,  and  when  injected  into  mice  in  small  amounts 
caused  a  local  subcutaneous  oedema  ending  in  recovery. 
Larger  doses  caused  more  extensive  oedema  and  death. 
A  fatal  dose  for  a  mouse  weighing  22  gm.  was  0.3 
gm.  Boiling  for  a  short  time  diminished  the  toxicity 
of  these  proteids  without  completely  destroj' ing  it.  The 
alkaloid  produced  similar  symptoms  when  injected  into 
mice  but  more  promptly  and  in  a  smaller  dose — 0.1  to 
0.15  gm.  killed  a  mouse  weighing  22  gm.  in  two  or  three 


ANTHRAX.  91 

hours.  Hankin  and  Westbrook  have  more  recently 
(1892)  made  researches  with  reference  to  the  proteids 
present  in  anthrax  cultures.  To  obtain  an  immunizing 
albumose  they  cultivated  the  bacillus  at  20°  C  in  flesh- 
extract  solution  (1  to  1,000)  to  which  fibrin  was  added. 
At  the  end  of  eight  days  a  considerable  precipitate  was 
obtained  by  means  of  ammonium  sulphate.  This  was 
placed  in  a  dialyzer  in  running  water  at  42^^  to  45^  C.  ; 
then  precipitated  by  alcohol  and  dissolved  in  a  small 
quantity  of  water  (30  c.c.) — 500  cc.  of  llesh-extract  treated 
in  this  way  gave  only  0.44  gm.  of  albumose.  Experi- 
ments on  mice  gave  some  evidence  of '  the  immunizing 
action  of  this  albumose,  but  the  results  were  apparently 
not  so  definite  as  those  previously  reported  by  Hankin 
(p.  89).  Nor  are  the  experiments  of  Petermann,  who 
followed  Haukin's  method  (1892),  more  satisfactory.  Ar- 
loing  obtained  more  favorable  results  by  using  culture 
liquids  from  which  the  bacilli  had  been  removed  by  sedi- 
mentation. A  considerable  i^recipitate  was  obtained 
when  alcohol  was  added  to  the  culture  liquid,  but  it  was 
found  that  this  precipitate  had  no  immunizing  effect. 
On  the  contrary,  there  remained  in  solution  an  immuniz- 
ing substance.  This  was  obtained  in  a  concentrated 
form  by  evaporating  at  50°  C.  in  a  partial  vacuum.  Ex- 
periments upon  lambs  showed  the  protective  power  of 
this  extract,  and  of  the  culture  liquids  before  treatment 
when  injected  in  considerable  quantity. 

In  a  paper  published  in  the  Fortschritte  der  irediein, 
AVysokoAvicz  gives  a  resume  of  the  results  obtained  in 
Russia  in  protective  inoculations  made  up  to  date  of 
publication  (January,  1889).  According  to  the  author 
named,  Professor  Cenkowski,  who  had  made  himself  fa- 
miliar with  Pasteur's  method  while  on  a  visit  to  Paris, 


92  IMMUNITY   AND    SERUM-THERAPY. 

was  the  first  to  employ  it  iu  Eussia  (1883).  But  he 
I'ouiul  its  aj)plicatiou  to  be  attended  with  some  difficul- 
ties. The  cultures  attenuated  as  directed  by  Pasteur  at 
42°  to  43°  C.  "  showed  a  very  different  degree  of  virulence 
in  different  experiments,  and  their  virulence  was  also 
changed  by  keeping."  Experiments  were  therefore  made 
with  a  view  to  securing  a  more  satisfactory  vaccine.  In 
an  experiment  made  iu  1885,  1,333  sheep  were  inocu- 
lated ;  of  these  21  died  from  the  first  inoculation  and  4 
from  the  second  (1.86  per  cent.).  Subsequenth"  better 
results  were  obtained,  and  up  to  the  end  of  1888,  20,310 
sheep  had  been  inoculated,  with  an  average  mortality  of 
0.87  per  cent,  as  a  result  of  the  inoculations. 

Professor  Cenkowski  found  that  greater  losses  occurred 
when  the  inoculations  were  made  in  midsummer  or  mid- 
winter than  when  they  were  made  in  the  spring  or  au- 
tumn. The  losses  from  anthrax  diminished  among  the 
flocks  in  which  the  protective  inoculations  were  prac- 
tised in  i^roportion  to  the  number  of  sheej)  inoculated, 
falling  from  8.3  per  cent,  in  1884,  the  year  before  the  in- 
oculations were  commenced,  to  0.13  per  cent,  in  1888. 
The  author  of  the  paper  states  thaf  in  some  parts  of 
Russia  the  annual  loss  among  the  sheep  from  anthrax  is 
as  high  as  thirty-three  per  cent. 

The  reliability  of  the  j^rotective  inoculations  was 
tested  by  a  Commission,  to  which  Wysokowicz  belonged. 
Fifty  sheep  which  had  been  inoculated  from  two  to  four 
months  previously  were  infected  with  virulent  anthrax 
material.  Of  these  one  only  died.  Later,  twenty  sheep 
which  had  been  inoculated  thirteen  months  before  were 
inoculated  with  virulent  material.  Of  these  two  died. 
These  favorable  results  are  ascribed  by  WysokoAvicz  to 
the    improved     method    of    attenuating    anthrax    virus 


ANTHRAX.  93 

adopted  by  Professor  Cenkowski.  As  a  first  vaccine  he 
employed  a  culture  whicli  was  stronger  than  that  of  Pas- 
teur, and  which  killed  mice  and  caused  the  death  of  one- 
third  of  the  Zieselmause  (Spermophilus  citillus)  inocu- 
lated. He  used  as  a  vaccine  an  attenuated  culture  which 
had  l)een  carried  through  a  series  of  the  animals  last 
mentioned.  His  vaccine,  consisting*  of  a  bouillon  cult- 
ure from  a  drop  of  blood  of  the  animal,  was  preserved  by 
the  addition  of  two  parts  of  a  thirty  per  cent,  solution  of 
pure  glycerine  to  one  part  of  the  culturQ. 

For  inoculating  a  sheep  of  average  size  he  used  0.1  to 
0.2  c.c.  of  this  first  vaccine,  for  a  larger  animal  from  0.3 
to  0.5  c.c.  This  second  inoculation  was  made  twelve 
days  after  the  first,  with  a  virus  which  killed  three-fourths 
of  the  Zieselmause  and  from  one-third  to  one-half  of  the 
rabbits  inoculated  with  it.  Numerous  experiments  con- 
vinced Cenkowski  that  no  change  occurred  in  the  viru- 
lence of  his  different 'vaccines  when  they  were  carried 
through  a  series  of  mice  or  of  earless  marmots  (Ziesel- 
mause). 

Hess  reports  that  the  anthrax  inoculations  made  by 
Chauveau's  method  in  the  Canton  Bern,  during  the  years 
1886,  1887,  and  1888,  were  not  attended  with  any  losses 
either  from  the  inoculations  or  from  subsequent  attacks 
of  anthrax  among  the  inoculated  animals  (cattle  ?).  In  all 
two  hundred  and  fifty-three  animals  were  inoculated  dur- 
ing the  three  years  specified. 

Hutyra  (1890)  has  reported  upon  anthrax  inoculations 
by  Pasteur's  method,  as  carried  out  under  the  regulations 
of  the  Government  in  1889.  The  number  of  horses  inoc- 
ulated was  130,  2  of  which  died  of  anthrax  at  a  later  date 
— not  as  a  result  of  the  inoculation.  This  gives  a  percent- 
age of  loss  of  1.35,  which  is  much  below  the  usual  rate 


94  IMMUNITY   AND   SERUM-THERAPY. 

without  protective  inoculations.  Three  thousand  two  hun- 
dred and  seventy -nine  cattle,  belong-ing-  to  tliirtj'-two  dif- 
ferent estates,  were  inoculated.  Of  these  11  died  from  an- 
thrax, and  2  of  these  as  a  result  of  the  first  inoculation. 
Deducting-  these  2  the  loss  was  0.27  per  cent.,  Avhereas  in 
former  years  the  losses  in  the  same  herds  had  been  from 
six  to  twelve  i^er  cent.  Twenty-two  thousand  seven  hun- 
dred and  sixty-seven  sheep  were  inoculated  on  twenty- 
three  different  estates.  One  hundred  and  sixty -two  of 
these  died  from  the  first  inoculation  and  59  within  twelve 
days  after  the  second  inoculation.  In  the  coarse  of  the 
year  432  of  the  inoculated  animals  died  from  anthrax — 
in  all  a  loss  of  2.18  per  cent.  In  the  absence  of  protective 
inoculations  the  annual  loss  in  these  flocks  had  been 
about  ten  per  cent.  It  was  found  that  lambs  four  months 
old  could  be  inoculated  with  the  same  dose  as  the  older 
sheep,  and  without  any  greater  loss  as  a  result  of  the 
operation. 

The  result  of  anthrax  inoculations  made  in  France  by 
Pasteur's  method  during  the  past  twelve  years  have  re- 
cently (1894)  been  summarized  by  Chamberland.  The 
veterinarians  who  made  the  inoculations  were  each  j'ear 
called  upon  to  answer  the  following  questions  :  1.  Num- 
ber of  animals  inoculated.  2.  Number  of  deaths  from 
first  inoculation.  3.  Number  of  animals  dying  within 
twelve  days  after  second  inoculation.  4.  Number  of  ani- 
mals dying  of  anthrax  within  a  year  after  protective  in- 
oculations. 5.  The  yearly  average  loss  before  inocula- 
tions were  practised.  The  total  number  of  animals 
inoculated  during  the  period  to  which  this  report  refers 
was  1,788,677  sheep  and  200,962  cattle.  The  average  an- 
nual loss  before  these  protective  inoculations  were  prac- 
tised is  said  to  have  been  about  ten  per  cent,  for  sheep 


ANTHRAX.  95 

and  five  per  cent,  for  cattle.  The  total  mortality  from 
this  disease  among  inoculated  animals,  including-  that 
resulting  from  the  inoculations,  was  0.94  per  cent,  for 
sheep  and  0.34  per  cent,  for  cattle.  Chamberland  esti- 
mates that  the  total  saving  as  a  result  of  tiie  inoculations 
practised  has  been  5,000,000  francs  for  sheep  and  2,000,000 
francs  for  cattle. 

Podmolinotf  gives  the  following  summary  of  results 
obtained  in  1892  and  1893  in  the  "Government  of  Cher- 
son  "  (Austria) :  Number  of  sheep  inoculated,  67,176 ; 
loss,  294,  =  0.43  per  cent.  Number  of  horses  inoculated, 
1,452  ;  loss,  8.  Number  of  cattle  inoculated,  3,652  ;  loss, 
2.  The  conclusion  is  reached  that  Pasteur's  method  of 
inoculation  affords  an  immunity  against  infection  with 
virulent  anthrax  bacilli  in  greater  amounts  than  could 
ever  occur  under  natural  conditions. 

Serum-therapy. 

The  Japanese  bacteriologists,  Ogata  and  Jasuhara, 
published  in  1890  their  paper  relating  to  the  modifica- 
tion of  virulence  in  anthrax  bacilli  by  cultivation  in  the 
blood-serum  of  immune  animals  and  the  therapeutic  use 
of  blood-serum  from  the  frog  or  dog  for  the  cure  of  an- 
thrax in  susceptible  animals  after  infection.  It  was  found 
that  an  anthrax  culture  which  killed  mice,  when  grown 
upon  blood-serum  from  immune  animals,  such  as  the 
frog,  the  rat,  or  the  dog,  became  attenuated  to  such  an 
extent  that  when  inoculated  into  mice  a  fatal  result  no 
longer  occurred.  But  more  surprising  was  the  announce- 
ment that  when  mice  received  a  drop  of  frog's  blood,  or 
half  a  drop  of  dog's  blood,  some  time  before  (as  much  as 
seventy-two  hours)  or  shortly  after  (five  hours)  infection 


96  IMMUNITY   AND   SERUM-THERAPY. 

with  anthrax  bacilli,  they  all  recovered  and  subsequently 
proved  to  be  immune.  When  the  blood  of  the  frog-,  dog, 
or  rat  was  subjected  to  a  temperature  of  45°  C.  for  an 
hour  it  no  longer  had  any  curative  or  immunizing"  action. 
Similar  experiments  are  said  to  have  been  made  upon 
rabbits  and  guinea-pigs  with  the  same  result.  Accord- 
ing to  the  authors  mentioned  the  proportion  of  dog's 
blood  required  to  protect  a  mouse  from  the  fatal  effects 
of  an  anthrax  inoculation  is  about  one  part  to  eight  hun- 
dred inxrts  of  body-weight.  But  these  remarkable  re- 
sults reported  by  the  Japanese  investigators  have  not 
been  confirmed  by  subsequent  researches.  Several  Italian 
bacteriologists,  who  repeated  the  experiments,  in  1891, 
failed  to  cure  infected  animals  with  considerably  larger 
doses  of  serum  from  immune  animals.  Thus  Pane  found 
that  the  blood-serum  of  dogs  and  pigeons  did  not  iDre- 
serve  guinea-pigs  from  anthrax  infection.  Bergonzini  in- 
jected blood-serum  of  the  dog  into  the  peritoneal  cavity 
of  guinea-pigs  in  doses  of  3.5  to  17  c.c.  without  preserv- 
ing them  from  fatal  infection,  no  matter  whether  the 
serum  was  injected  before  or  after  or  at  the  same  time  as 
the  anthrax  virus.  Serafini  and  Enriquez  (1891)  also 
failed  entirely  to  j)revent  a  fatal  result  after  anthrax  in- 
fection by  injecting  serum  from  immune  animals  into  the 
animal  being  experimented  upon.  Their  experiments 
were  made  upon  22  rabbits,  43  guinea-pigs,  3  white  mice, 
and  38  gray  mice,  all  of  which  died  of  anthrax.  The 
quantit}^  of  serum  or  blood  used  varied  from  one  drop  to 
25  c.c,  and  three  rabbits  received  several  doses  of  dog 
serum  of  40  c.c.  each.  Injections  were  made  subcuta- 
neously,  or  into  the  peritoneal  cavity,  and  in  some  in- 
stances into  the  circulation  through  a  vein.  The  injec- 
tion of  a  mixture  of  1  ose  of  an  anthrax  culture  with  5 


ANTHRAX.  97 

c.c.  of  blood-serum  caused  the  death  of  the  inoculated 
animal. 

BIBLIOGRAPHY. 

Arloing,  S.  :  Sur  la  presence  et  la  nature  de  la  substance  phylacogene 

dans  les  cultures  liquides  ordinaires  du  bacillus  antliracis.    Compte- 

reudu,  Acad,  des  Sci.,  cxiv.,  1521,  1892, 
Rechercbes  sur  la  vaccination  par  les  produits  solubles  contenus 

dans  les  cultures  du  bacillus  anthracis.     Journal  de  Med.  veterin. 

et  zootechn.,  1892,  p.  505. 
Bebring  :  Ueber  die  Ursacbe  der  Immunitat  von  weissen  Ratten  gegen 

Milzbrand.     Centralbl.  fur  klin.  Med.,  1888,  No.  38. 
Bergonzini :  Sull'  azione  preventiva  contro  il  carboncliio  dello  siero  di 

sangue  di  animali  immuni.     Rass.  di  Sci.  Med.,  1891,  No.  10. 
Chamberland,  Ch.  :    Resultats  pratiques    des   vaccinations    contre  le 

charbon  et  le  rouget  en  France.     Annales  de  I'lnst.  Pasteur,  1894, 

p.  161. 
Czaplewski,  E.  :  Weitere  Untersuchungen  uber  die  Immunitat  der  Tau- 

ben  gegen  Milzbrand.     Zeitschr.  fiir  Hyg.,  XII.,  348,  1892. 
Dungern,  Freilierr  v.  :    Ueber  die  Hemmung  der  Milzbrandinfektion 

durcb  Friedlander'scbe  Bakterien  im  Kanincbenorganismus.     Zeit- 

scbrift  f.  Hygiene,  XVIII.,  1894. 
Emmericb,  R.  :  Die  Heilung  des  Milzbrandes.    Arcbiv.  fur  Hygiene,  Bd. 

VI.,  1886,  p.  442. 
Emmericb  und  de  Matlei  :  Die  Vernicbtung  der  Milzbrandbacillen  im 

Organismus.     Fortscbr.  der  Medicin,  1887,  No.  20. 
Gameleia  :  Etude  sur  la  vaccination  cbarbonneuse.     Annales  de  I'lnst. 

Pasteur,  1888,  p.  517. 
llankin,  E.,  et  Westbrook,*  F.  F.  :  Sur  les  albumoses  et  les  toxalbumines 

secretees  par  le  bacille  cbarbonneux.     Annales  de  I'lnst.  Pasteur, 

vi.,  633,  1892. 
Hutyra :    Scbutzimpfungen  gegen  Milzbrand  in  Ungarn.      Ungarn's 

Veterinar-Bericbte  fiir  1891. 
Koch  :  Ueber  die  Milzbrandimpfung.     Eine  Entgegnung  auf  den  von 

Pasteur  in  Genf  gebaltenen  Vortrag.      Kassel  and  Berlin,  1882, 

pp.  37. 
Lazarus,  A.,  und  Weyl,  Tb.:  Weitere  Beilrage  zur  Tbeoric  der  Immuni- 
tat gegen  Milzbrand.    Berl.  klin.  Wocbenschr.,  1892,  No.  45. 
Neuscbtube,  S.  F.  :  Die  propbylaktiscben  Milzbrandimpfungen  beim 

Scbafe  und  bei  anderen  Haustieren  nach  der  Metbode  von  Prof. 

Zenkowsky.     8°,  53  p.     St.  Petersburg,  1893  (Russian). 
Ogata  and  Tasubara  :  Op.  cit.,  p.  69. 
Pane,  N.  :  Ueber  die  Immunisierung  der  Kanincben  gegen  das  Virus 

des  Milzbrandes,  etc.    Abst.  in  Centralbl.  fiir  Bakteriol.,  XVI. ,  346. 

1894. 

7 


98  IMMUNITY    AN'D   SERUM-TIT  EKAPY. 

Pasteur  :    Reclierches  sur  I'etiologie  et  la  propliylaxie  de  ];i  maladie 

charbonneuse  dans  le  depurtemeut  d'Eure-et-Loiie.     Rec.  de  Med. 

vet.,  Paris,  1879,  p.  193. 
Nouvelles  observations    sur    I'etiologie    et   la  prophylaxie   du 

charbou.     Corapte-rendu,  Acad,  des  Sci.,  1880,  xci.,  ]>.  G97. 
Resultats  des  vaccinations  charbonneuses  pratiques  pendant  les 

mois  de  juillet,  aout,  et  septembre,  1881.     Arch,  vet.,  Paris,  vii. , 

1882,  p.  177. 
De  la  possibilite  de  rendrc  les  moutons  refractaires  au  charbon 

par  la  methode  des  inoculations  preventives  :  avec  la  collaboration 

de  MM.  Cbamberland  et  Roux.      Conipte-rendu,  Acad,  des  Sci., 

xcii.,  1881,  pp.  662,  665. 

Une  statistique  au  sujet  de  la  vaccination  preventive  centre  le 


charbon,  portant  sur  quatre-vingt-ciuci  mille  animaux.    Ibid.,  xcv., 

1882,  p.  1250. 
Perroucito  :  Studien  iiber  Immunitiit  gegen  Milzbrand.     Centralbl.  fiir 

Bakteriol.,  Bd.  V.,  1889,  p.  50,3. 
Petermann  :   Recherches  sur  I'immunite  contre  "le  charbon.     Annales 

de  I'Inst.  Pasteur,  t.  vi.,  1892,  p.  32. 
Podmolinoff  :  Bericht  iiber  die  in  1893  im  Chersonlschen  Gouvernment 

ausgefiihrten  Milzbi-andschutzimpfungen.     Arch.  f.  Wissencli.  u. 

Prakt.  Tierheilk.,  1894,  p.  805. 
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lato  ad  animali  suscettibili  pel  carbonchio.     Riforma  Med.,  1891, 

No.  152. 
Toussaint :  De  I'immunite  pour  le  charbon,  acquise  A  la  suite  d'inoc- 

iilations  preventives.     Compte-rendu,  Acad,  des  Sci.,  xci.,  1880,  p. 

135. 
Weyl,  Th.  :  Zur  Tlieorie  der  Immunitiit  gegen  Milzbrand.     Zeitschr.  f^ 

Hygiene,  XL,  881,  1892. 
Wyssokowitsch  :  Ueber  Schutzimpfung  gegen  Milzbrand  in  Russlando 

Fortschr.  der  Med.  1889,  p.  1. 


II. 

CHICKEN  CHOLERA. 

Pasteur's  researches  with  reference  to  the  etiolog-y  of 
the  disease  known  in  France  as  cholera  de-'^  2)otdcs,  first  led 
him  to  the  discovery  that  a  virnlent  culture  of  a  jiatho- 
genic  bacterium  may  become  "  attenuated  "  by  certain 
agencies,  and  that  immunity  may  be  conferred  upon  sus- 
ceptible animals  by  inoculating  them  with  such  attenu- 
ated culture.  We  now  know  that  his  microbe  of  fowl 
cholera  is  a  widely  distributed  bacillus,  which  is  fre- 
quently encountered  in  putrefying  material,  and  that  it 
is  also  extremely  fatal  to  pigeons,  pheasants,  sparrows, 
rabbits,  and  mice.  Also  that  the  same,  or  nearly  allied 
species,  may  produce  an  infectious  disease  of  swine 
(Schweineseuche),  of  cattle  {R'uiderseucJie),  and  of  deer 
( Wildseuche). 

Subcutaneous  injection  of  a  minute  quantity  of  a  viru- 
lent culture  usually  kills  chickens  within  forty-eight 
hours.  Some  time  before  deatli  the  fowl  falls  into  a 
somnolent  condition,  and,  with  drooping  wings  and  ruf- 
fled feathers,  remains  standing  in  one  place  until  it  dies. 
Infection  may  also  occur  from  the  ingestion  of  food 
moistened  with  a  culture  of  the  bacillus,  or  soiled  with 
the  discharges  from  the  bowels  of  other  infected  fowls. 
At  the  autopsy  the  mucous  membrane  of  the  small  intes- 
tine is  found  to  be  inflamed  and  studded  with  small 
hemorrhagic  foci,  as  are  also  the  serous  membranes  ;  the 


100  IMMUNITY   AND   SERUM-THERAPY. 

spleen  is  notably  enlarged.  The  bacilli  are  found  in 
great  numbers  in  the  blood,  in  the  various  organs,  and  in 
the  contents  of  the  intestine.  In  rabbits  death  commonly 
occurs  in  from  sixteen  to  twenty  hours,  and  is  often  pre- 
ceded by  convulsions.  The  temperature  is  elevated  at 
first,  but  shortly  before  death  it  is  reduced  below  the 
normal.  The  post-mortem  appearances  are  :  Swelling  of 
the  spleen  and  lymphatic  glands  ;  ecchymoses  or  diffuse 
hemorrhagic  infiltrations  of  the  mucous  membranes  of 
the  digestive  and  respiratory  passages,  and  in  the  mus- 
cles ;  and  at  the  point  of  inoculation  a  slight  amount  of 
inflammatory  oedema.  The  bacilli  are  found  in  consider- 
able numbers  in  the  blood  within  the  vessels,  or  in  that 
which  has  escaped  into  the  tissues  by  the  rupture  of 
small  veins.  They  are  not,  however,  so  numerous  as  in 
some  other  forms  of  septicaemia — e.g.^  anthrax,  mouse  sep- 
ticaemia— when  an  examination  is  made  immediately  after 
death ;  later,  the  number  may  be  greatly  increased  as  a 
result  of  post-mortem  multiplication  within  the  vessels. 
The  rabbit  is  so  extremely  susceptible  to  infection  by  this 
bacillus  that  inoculation  in  the  cornea  by  a  slight  super- 
ficial wound  usually  gives  rise  to  general  infection  and 
death.  This  animal  may  also  be  infected  by  the  inges- 
tion of  food  contaminated  wdth  a  culture  of  the  bacillus. 
It  is  by  this  means  that  Pasteur  proposed  to  destroy  the 
rabbits  in  Australia,  which  have  multiplied  in  that  coun- 
try to  such  an  extent  as  to  constitute  a  veritable  pest. 
Both  in  fowls  and  in  rabbits  the  disease  may,  under  cer- 
tain circumstances,  run  a  more  protracted  course — e.g., 
when  they  are  inoculated  with  a  small  quantity  of  an  at- 
tenuated culture.  In  less  susceptible  animals — guinea- 
pigs,  sheep,  dogs,  horses — a  local  abscess,  without  general 
infection,  may  result  from  the  subcutaneous  injection  of 


CHICKEN    CHOLERA.  101 

the  bacillus  ;  but  these  animals  are  not  entirely  immune. 
In  the  infectious  maladies  of  swine,  cattle,  deer,  and  other 
large  animals,  to  which  reference  has  been  made,  and 
which  are  believed  to  be  due  to  the  same  bacillus,  the 
symptoms  and  pathological  appearances  do  not  entirely 
correspond  with  those  in  the  rabbit  or  the  fowl ;  but  the 
bacillus  as  obtained  from  the  blood  of  such  animals  cor- 
responds in  its  morphological  and  biological  characters 
with  Pasteur's  microbe  of  fowl  cholera,  and  Koch's  bacil- 
lus of  rabbit  septicaemia,  and  pure  cultures  from  the  vari- 
ous sources  mentioned,  are  equally  fatal  to  rabbits  and  to 
fowls.  In  the  larger  animals  pulmonary  and  intestinal 
lesions  are  developed,  and  in  swine,  a  diffused  red  color 
of  the  skin,  similar  to  that  observed  in  the  disease  known 
in  Germany  as  Schweinerothlauf  (Fr.  rouget),  is  some- 
times seen. 

According  to  Baumgarten  bacilli  from  Wildseuche  Or 
from  Rlnde7'seuche  inoculated  into  swine  give  rise  to  fatal 
Sclmveineseuche,  and  bacilli  from  any  of  these  forms  of  dis- 
ease, when  inoculated  into  pigeons,  produce  character- 
istic fowl  cholera;  but 'the  bacillus  as  obtained  from 
Scfiioeineseiiche  or  Wildseuche  is  not  fatal  to  chickens,  and 
the  bacillus  from  Schimineseuche  is  fatal  to  guinea-pigs, 
which  have  but  slight  susceptibility  to  the  bacillus  of 
rabbit  septicaemia.  Notwithstanding  these  differences 
he  agrees  with  Hueppe  in  the  view  that  the  bacilli  from 
the  various  sources  mentioned  are  specifically  identical ; 
although  evidently,  if  this  view  is  adopted,  we  must  ad- 
mit that  varieties  exist,  which  differ  somewhat  in  their 
pathogenic  power. 

In  the  writer's  "  Manual  of  Bacteriology  "  this  bacillus 
is  described  under  the  name  Bacillus  septicmmice  hemor- 
rhagicce,  first  proposed  for  it  by  Hueppe.     In  the  present 


102  I]MM UNITY   AND   SERUM-THERAPY. 

cbaiDter  we  shall  give  an  account  of  the  exiaerimental  evi- 
dence relating-  to  protective  inoculations  and  serum  ther- 
apy in  various  animals,  with  the  different  varieties  of  the 
bacillus  in  question  which  have  been  encountered. 

It  seems  probable  that  the  same  bacillus  was  the  cause 
of  the  fatal  form  of  septicaemia  studied  by  Davaine,  and 
which  resulted  from  the  inoculation  of  susceptible  ani- 
mals with  putrefying  blood.  These  experiments  by  the 
distinguished  French  physician  constitute  an  important 
part  of  the  pioneer  work  in  this  field  of  research.  They 
were  commenced  in  1868,  and  are  x>ublished  in  the  Bul- 
letin of  the  Academy  of  Medicine  {seance  of  February  18, 
1879). 

Davaine,  in  the  paper  referred  to,  calls  attention  to  the 
fact,  developed  by  his  experiments,  that  there  is  a  great 
difference  in  the  resisting  iDower  of  different  animals  to 
nie  form  of  septicaemia  which  had  been  the  subject  of  his 
investigations.  Thus  the  rabbit  succumbed  when  inocu- 
lated with  a  millionth  part  of  a  drop  of  blood,  while 
guinea-pigs  and  dogs  remained  unaffected  by  such  small 
doses.  With  reference  to  the  specific  cause  of  the  form 
of  septicaemia  discovered  by  him,  Davaine  says  : 

"  The  virus  is  one  of  the  bacteria  of  putrefaction.  I 
say  '  one  of  the  bacteria '  because  there  is  reason  to  be- 
lieve that  there  are  among  these  minute  organisms  nu- 
merous species  which  do  not  all  develop  at  the  same  time 
w^hen  they  are  present  in  various  media." 

Davaine  also  discovered  the  fact  that  infection  de- 
pends, within  certain  limits,  upon  the  quantity  of  bacte- 
ria introduced  into  the  tissues.     He  says  : 

"  This  question  of  quantity  was  manifest  in  our  exper- 
iments.    Not  only   did  it  vary  in  different  species,  the 


CHICKEN   CHOLERA.  103 

rabbit  and  the  dog  for  example,  but  it  may  vary  iu  the 
same  species," 

The  identity  of  "  Davaine's  septicaemia  "  with  Pasteur's 
choUra  des  ponies  is  made  still  more  probable  by  the  ex- 
perimental evidence  offered  by  Toussaint  iu  a  communi- 
cation to  the  French  Academy  of  Sciences,  made  by  M. 
Bouley  at  the  seance  of  July  25,  1881.  In  this  communi- 
cation Toussaint  says  : 

"Three  years  ago,  July  8,  1878,1  had  the  honor  to 
present  to  the  Academy  an  account  of  a  malady  due  to 
microbes,  which  I  identified  with  that  studied  by  Da- 
vaine  in  18G4  and  1865,  and  which  he  differentiated  from 
anthrax,  for  which  it  had  been  mistaken  by  Leplat  and 
Jaillard. 

"  In  the  month  of  December,  1878,  I  made  acquaint- 
ance with  fowl  cholera,  and  already,  in  my  thoughts,  1 
identified  this  disease  with  that  which  I  had  observed  in 
\\\y  experiments  made  early  in  the  year.  The  microbes 
of  the  two  diseases  resembled  each  other  perfectly  and 
behaved  the  same  when  inoculated  in  rabbits.  I  had, 
even  in  1879,  sent  to  M.  Bouley  tAvo  notes,  in  which  I 
called  attention  to  the  analogies  which  exist  between  the 
parasites  of  the  two  diseases  and  the  lesions  which  they 
determine,  not  only  in  the  rabbit  but  also  in  pigeons  and 
fowls. 

"  The  experiments  of  the  same  kind  made  at  the  end  of 
1879  and  in  1880  caused  me  to  insert  the  note  published 
on  page  301,  vol.  xci.,  of  the  Convptes-remlvs,  under  the  title 
of  :  '  Identity  of  Acute  Experimental  Septicaemia  and 
Fowl  Cholera.'  I  gave  a  resiwie  in  this  note  of  five  series 
of  experiments  which  had  demonstrated  to  me  that  inoc- 
ulations of  the  microbe  of  septicaemia  give  rise  to  the 
manifestations  of  fowl  cholera.  These  results  have  re- 
cently been  confirmed  by  additional  facts." 

Toussaint  closes  his  paper  by  some  remarks  upon  the 
origin  of  epidemics  of  fowl  cholera  which  we  quote  be- 


104  IMMUNITY   AND   SERUM-TIIEnAPY. 

cause  we  believe  that  the  additions  made  to  our  knowl- 
edge of  the  microbe  which  causes  this  disease  give  sup- 
port to  the  views  advanced  by  him  in  1881 : 

"  The  causes  which  determine  epidemics  of  fowl  cholera 
are  yet  unknown.  It  has  been  supposed  that  putrefactive 
substances  may  give  rise  to  them,  and  this  has  led  to  the 
recommendation  of  cleanliness  and  disinfection  for  their 
prevention.  The  microbe  which  kills  the  first  fowl  in  an 
epidemic  certainly  came  from  some  anterior  generation 
which  had  killed  others.  But  how  was  it  perpetuated  ? 
Do  not  the  facts  which  demonstrate  the  development  of 
septicfemia  from  material  undergoing  putrefaction  throw 
some  light  on  the  question  of  etiology  ?  Is  it  not  prob- 
able that  the  fowls  find  the  conditions  of  infection  with 
cholera  in  the  presence  of  organic  matter  undergoing  pu- 
trefaction, which  may  serve  as  a  culture  medium  for  the 
germs  of  septicoemia  which  are  in  suspension  in  the  air 
together  with  the  ordinary  germs  of  putrefaction  ?  " 

Pasteur's  first  communication  relating  to  the  etiology 
of  fowl  cholera  was  made  to  the  French  Academy  at  the 
seance  of  February  9,  1880.  In  this  communication  he 
calls  attention  to  the  fact  that  when  fowls  are  fed  with 
bread  or  meat  soiled  with  a  small  quantity  of  a  culture  of 
the  microbe  of  fowl  cholera  they  become  infected  and 
their  discharges  contain  the  bacillus  in  large  numbers,  a 
fact  which  readily  accounts  for  the  sjjread  of  the  disease 
in  a  poultry -yard  when  a  case  occurs. 

In  the  same  communication  Pasteur  records  his  obser- 
vation that  "  by  a  certain  change  in  the  method  of  culti- 
vation the  infectious  microbe  may  be  caused  to  have  a 
diminished  virulence."  Also  the  fact  that  fowls  inocu- 
lated with  this  "  attenuated  "  virus  recover  and  are  subse- 
quently immune  against  infection  by  the  most  virulent  mi- 
crobes.   In  concluding  this  communication  Pasteur  sa3's : 


CHICKEN    CHOLERA.  105 

"  It  appears  to  be  superfluous  to  point  out  the  princi- 
pal result  of  the  facts  which  I  have  had  the  honor  to  pre- 
sent to  the  Academy.  There  are  two,  however,  w^hich  it 
may  be  useful  to  mention.  These  are,  first,  the  hope  of 
obtaining  artificial  cultures  of  all  kinds  of  virus ;  second, 
the  idea  of  seeking-  for  virus  vaccines  of  the  virulent  mal- 
adies which  have  devastated  so  often,  and  still  devastate, 
the  human  race,  and  w^hich  are  such  a  scourge  to  that 
branch  of  agriculture  which  relates  to  the  breeding  of 
domestic  animals." 

In  his  communication  of  October  26,  1880,  Pasteur 
gives  his  reasons  for  concluding  that  attenuation  of  viru- 
lence is  due  to  the  action  upon  the  microbe  of  atmos- 
pheric oxygen.  He  infers  this  from  the  fact,  demon- 
strated by  experiment,  that  when  cultures  are  placed  in 
hermetically  sealed  tubes,  from  which  the  oxygen  present 
is  soon  exhausted  by  the  growth  of  the  microbe,  they  do 
not  become  attenuated  in  virulence ;  whereas  cultures 
which  are  freely  exposed  to  the  air  gradually  become 
attenuated.  Pasteur  sees  in  this  an  impoi-tant  fact  bear- 
ing upon  the  explanation  of  the  natural  extinction  of 
epidemics.     He  says : 

^'  May  we  not  sujDpose,  then,  that  it  is  to  this  influence 
that  we  must  attribute,  in  the  present  as  in  the  past,  the 
limitation  of  great  epidemics." 

In  his  communication  to  the  French  Academy,  made 
on  February  28,  1881,  Pasteur  treats  of  the  attenuation  of 
virulence  by  the  method  above  referred  to  and  by  the 
method  of  Toussaint,  and  also  of  the  re-establishment  of 
the  virulence  of  attenuated  cultures.     He  says  : 

"  The  secret  of  the  return  to  virulence  rests  solely,  at 
present,  upon  successive  cultures  in  the  bodies  of  certain 
animals." 


106  IMMUNITY   AND   SEKUM-TIIERAPY. 

Thus  lie  had  found  by  experiment  that  the  anthrax  ba- 
cillus might  be  so  attenuated  that  it  was  harmless  for 
grown  guinea-pigs,  or  even  for  guinea-pigs  a  month  or  a 
week  old,  but  it  would  still  kill  guinea-pigs  just  born — a 
day  old.  By  inoculating  an  older  pig  with  the  blood  of 
this  one,  and  so  on,  the  virulence  was  gradually  aug- 
mented, until  finally  a  virus  might  be  obtained  which 
would  kill  adult  animals,  and  even  sheep.  In  the  same 
way  the  attenuated  microbe  of  fowl  cholera  could  be  re- 
stored to  virulence  by  first  inoculating  small  birds,  such 
as  sparrows  or  canaries. 

Applying  these  facts,  demonstrated  by  his  experiments, 
to  the  explanation  of  the  origin  of  epidemics,  Pasteur 
says : 

"  I  finished  my  communication  on  October  26th  by 
calling  attention  to  the  attenuation  of  viruses  by  expos- 
ure to  the  air  as  being  probably  one  of  the  factors  in  the 
extinction  of  great  epidemics.  The  facts  presented  in  this 
paper,  in  their  turn,  may  serve  to  explain  the  so-called 
'  spontaneous  development '  of  these  scourges. 

"  An  epidemic  which  has  been  extinguished  by  the 
attenuation  of  its  virus  may  be  reborn  by  the  reinfovce- 
ment  of  this  virus  under  certain  influences.  The  accounts 
which  I  have  read  of  the  spontaneous  appearance  of  the 
plague  appear  to  me  to  oft'er  examples  of  this  ;  for  exam- 
ple, the  plague  at  Benghazi,  in  1856-58,  the  outbreak  of 
which  could  not  be  traced.  The  plague  is  a  virulent 
malady  which  belongs  to  certain  countries.  In  all  of 
these  countries  its  attenuated  virus  ought  to  exist,  ready 
to  resume  its  active  form  when  conditions  as  to  climate, 
famine,  and  distress  again  occur.  There  are  other  viru- 
lent maladies  which  appear  'spontaneously  '  in  all  coun- 
tries ;  such  as  camp  typhoid.  Without  doubt  the  germs 
of  the  microbes  which  cause  these  last-mentioned  mal- 
adies  are   everywhere    distributed.     Man    carries    them 


CHICKEN   CHOLERA.  107 

upon  him  or  in  liis  intestinal  canal  without  great  damage, 
but  ready  to  become  dangerous,  Avhen,  owing  to  constipa- 
tion or  to  successive  development  upon  the  surface  of 
wounds,  in  bodies  enfeebled  or  otherwise,  their  virulence 
is  progressively  reinforced." 

We  believe  that  the  more  complete  our  knowledg"e  re- 
lating to  the  origin  and  extinction  of  epidemics,  of  the 
kind  referred  to  by  Pasteur,  becomes,  the  more  apparent 
will  be  the  value  of  his  inductions  and  the  clearness  of 
his  scientific  foresight. 

Toussaint,  on  July  25,  1881,  reported  the  results  of  his 
experiments  upon  protecting  fowls  by  a  "  new  method  of 
vaccination."  This  consisted  in  inoculating  them  with 
the  blood  of  a  rabbit  which  had  recently  died  from  septi- 
caemia produced  by  the  same  microbe.  As  a  result  of 
such  inoculations  the  fowls  had  slight  local  lesions  at 
the  point  of  inoculation,  and  soon  recovered.  They  were 
subsequently  found  to  be  immune.  Cultures  from  the 
blood  of  a  septicBemic  rabbit  were  found  to  act  in  the 
same  way.  When  the  culture  had  been  passed  throug-h  a 
pigeon,  and  had  then  killed  a  fowl,  according-  to  Tous- 
saint, it  jDreserved  its  virulence  when  subsequently  passed 
through  the  rabbit. 

Salmon,  in  the  "Keiiort  of  the  Commissioner  of  Ag^ri- 
culture  "  for  1881  and  1882,  gives  an  account  of  his  exper- 
iments in  producing-  immunity  by  the  use  of  a  diluted 
virus.     He  says  : 

"  The  experiments  of  Chauveau,  taken  with  my  own, 
indicate  that  this  method  is  capable  of  generalization  to 
the  same  extent  as  that  discovered  by  Pasteur  ;  while  the 
ease  and  quickness  with  which  the  vaccine  is  prepared, 
the  certainty  of  effects,  the  economy  of  material,  and  the 
more  perfect  protection  are  points  which  would  appear 


108  IMMUNITY    AND   SERUM-THERAPY. 

to  make  it  decidedly  superior.  Wherever  the  cholera  of 
fowls  is  raging-  a  standard  cultivation  may  be  made  and 
the  vaccine  obtained  within  twenty-four  hours  ;  a  single 
drop  of  such  a  cultivation  will  vaccinate  ten,  twenty,  or 
even  forty  thousand  fowls,  and  within  three  weeks  of  the 
commencement  of  the  work  the  most  susceptible  of  our 
fowls  are  insusceptible  to  inoculations  with  the  strong- 
est virus.  And  this,  without  any  sickness,  or  even  local 
necroses,  which  Pasteur  describes  as  following  vaccina- 
tions with  his  attenuated  virus." 

In  discussing  the  practical  value  of  this  method  Sal- 
mon estimates  the  cost  as  trifling — "  not  more  than  half  a 
day's  time  of  one  man  for  one  hundred  fowls,  even  if 
three  inoculations  were  made." 

In  a  paper  on  protective  inoculations  against  fowl  chol- 
era, by  Kitt,  in  the  Deidsclte  Zeitschrift  fi'ir  Thiermedichi 
(December  20,  1886),  the  conclusion  is  reached  that  these 
inoculations  'undoubtedly  protect  the  fowls  from  infec- 
tion either  in  the  natural  way  or  by  inoculations  with 
virulent  material.  But  Kitt  doubts  the  practical  utility 
of  the  method  for  the  arrest  of  epidemics  of  this  disease 
in  the  poultry -yard ;  and,  as  we  think,  with  justice,  pre- 
fers to  depend  upon  cleanliness,  disinfection,  and  prompt 
removal  of  infected  fowls.  As  he  points  out,  a  consider- 
able time  is  required  to  produce  complete  immunity,  and 
two  inoculations  are  often  insufficient.  Pasteur  had  pre- 
viously reported  that  a  third  inoculation  is  usually  re- 
quired. But  the  infection  spreads  so  rapidly  when  an 
epidemic  is  developed  in  a  poultry-yard  that  a  large  pro- 
portion of  the  fowls  would  be  likely  to  perish  before  the 
protective  inoculations  could  be  carried  out.  Another 
objection  is  that  when  inoculated  in  the  breast  muscle 
the  value  of  the  fowl  for  the  table  is  reduced,  and  when 
inoculated  in  the  wing  an  unpleasant-looking  scab  is  left 


CHICKEN   CHOLERA.  109 

at  the  point  of  inoculation.  The  cost  in  material  and 
time  required  to  carry  out  the  three  successive  inocula- 
tions is  also  an  objection  to  the  practical,  application  of 
the  method.  Moreover,  the  excreta  of  the  inoculated 
fowls  contain  the  pathogenic  microbe,  and  it  would  evi- 
dently be  unwise  to  practise  inoculations  in  poultry- 
yards  not  already  infected.  Kitt  states,  also,  that  he  has 
always  succeeded  in  stamping-  out  the  disease  very 
promptly  by  the  other  measures  referred  to — disinfec- 
tion, cleanliness,  separation  of  all  fowls  which  show  any 
indications  of  being-  infected. 

In  a  more  recent  paper  (1893)  Kitt  reports  his  success 
in  conferring-  immunity  upon  fowls  by  a  new  method, 
which  is,  however,  rather  of  scientific  interest  than  of 
practical  value.  He  first  exiierimented  to  see  whether 
the  blood-serum  or  tissue  juices  of  immune  fowls  would 
g-ive  immunity  against  cholera  to  other  fowls,  and  ob- 
tained a  successful  result.  He  was  not,  however,  able  to 
produce  immunity  in  pigeons  or  in  rabbits  by  the  same 
method.  He  next  undertook  to  determine  whether  the 
immunizing  substance  was  present  in  the  eggs  of  fowls 
which  had  an  immunity  as  a  result  of  protective  inocula- 
tions. The  albumin  and  yolk  of  the  egg,  in  doses  of  5  to 
10  c.c,  was  injected  into  the  breast  of  fowls,  and  at  the 
end  of  ten  days  a  second  inoculation  of  the  same  kind 
was  made.  Six  days  after  the  second  inoculation  the 
fowls  (five)  and  a  control  hen  were  inoculated  with  viru- 
lent blood  from  a  pigeon,  and  at  the  same  time  fed  with 
the  chopped-up  fiesh  and  liver  of  a  pigeon  just  dead  from 
fowl  cholera.  The  control  hen  died  on  the  following  day 
from  typical  cholera,  the  others  remained  in  perfect 
health. 


no  IMMUNITY   AND   SERUM-THEKAPY 


BIBLIOGRAPHY. 

Gaffky  :  Die  Geflugelcliolera.   Zusammenfassender  Bericht.    Centralbl. 

fill-  Bakteriol.,  Bd.,  L,  1887,  p.  305. 
Kitt  :  Beitrage  zur  Kenntniss  derGefliigelclioleraund  deren  Scliutzimp- 

fling.     Deutsche  Zeitsclir.  fiir  Tliiermed.  iind  vergl.  Pathol.,  Bd. 

XIII.,  1886. 
Eine  neue  Schutzlmpfunggegen  Gefliigelpest.    (Gefliigelcholera.) 

Mtsh.  f.  prakt.  Thierheilk.,  IV.,  p.  59. 
Pasteur,  De  I'attenuation  du  virus  du  cholera  des  poules.     Compte- 

rendu,  Acad,  des  Sci.,  xci.,  p.  673. 


III. 

CHOLERA. 

The  spirillum  discovered  by  Koch  in  1884  is  now  pretty 
generally  recognized  as  the  specific  cause  of  Asiatic  chol- 
era. But  recent  researches  indicate  that  there  are  nu- 
merous pathogenic  varieties  of  this  spirillum,  and  show 
that  either  an  attenuated  cholera  spirillum  or  a  closely 
allied  saprophyte  is  not  infrequently  found  in  the  water 
of  rivers  in  various  parts  of  Europe.  As  this  spirillum  is 
found  in  the  intestine  of  cholera  patients,  and  not  in  the 
blood,  it  is  evident  that  its  pathogenic  action  depends 
upon  the  chemical  products  developed  during  its  growth, 
and  this  inference  is  fully  justified  by  the  results  of 
experiments  upon  the  loAver  animals.  These  chemical 
products  have  been  studied  by  Brieger,  Pfeiffer,  Sclioll, 
Gamaleia,  Westbrook,  and  others. 

Brieger  (1887)  succeeded  in  isolating  several  toxic 
ptomaines  from  cultures  of  the  cholera  spirillum,  some  of 
which  had  previously  been  obtained  from  other  sources 
— cadaverin,  putrescin,  creatinin,  methyl-guyanidin.  In 
addition  to  these  he  obtained  two  toxic  substances  not 
previously  known.  One  of  these  is  a  diamin,  resembling 
trimethyldiamin  ;  it  gave  rise  to  cramps  and  muscular 
tremor  in  inoculated  animals.  The  other  poison  reduced 
the  frequency  of  the  heart's  action  and  the  temperature 
of  the  body  in  the  animals  subjected  to  experiment.  In 
more  recent  researches  made  by  Brieger  and  Frankel 


112  IMMUNITY   AND   SERUM-TIIERAPY. 

(1890),  a  toxalbumin  was  obtained  from  cholera  cultures 
which,  when  injected  subcutaneously  into  guinea-pigs 
caused  their  death  in  two  or  three  days,  but  had  no  effect 
upon  rabbits. 

Pfeiffer  has  more  recently  (1892)  published  his  ex- 
tended researches  relating  to  the  cholera  poison.  He 
finds  that  recent  aerobic  cultures  of  the  cholera  spirillum 
contain  a  specific  toxic  substance  which  is  fatal  to  guinea- 
pigs  in  extremely  small  doses.  This  substance  stands  in 
close  relation  to  the  bacterial  cells,  and  is  perhaps  an  in- 
tegral part  of  the  same.  The  spirilla  may  be  killed  by 
chloroform,  thymol,  or  by  desiccation,  without  apparent 
injury  to  the  toxic  potency  of  this  substance.  It  is  de- 
stroyed, however,  by  absolute  alcohol,  by  concentrated 
solutions  of  neutral  salts,  and  by  the  boiling  tempera- 
ture, and  secondary  toxic  products  are  formed  which  have 
a  similar  pathogenic  action  but  are  from  ten  to  twenty 
times  less  potent.  Similar  toxic  products  were  obtained 
by  Pfeiffer  from  cultures  of  the  Finkler-Prior  spirillum 
and  from  spirillum  Metchnikovi. 

SchoU  (1890)  took  advantage  of  the  fact,  previously  de- 
monstrated by  Hueppe,  that  cultures  of  the  cholera  spiril- 
lum in  egg-albumin,  in  the  absence  of  oxygen,  are  more 
toxic  than  ordinary  bouillon  cultures.  Cultures  were  made 
by  Hueppe's  method  in  hen's  eggs.  No  poisonous  pto- 
maines were  found,  but  two  toxic  albuminous  substances 
were  obtained.  The  albuminous  liquid  from  the  egg  cult- 
ures was  dropped  into  ten  times  its  volume  of  absolute 
alcohol,  which  caused  a  white  precipitate,  a  portion  of 
which  sank  to  the  bottom  while  another  portion  floated  on 
the  surface.  The  portion  which  floated  was  easily  dissolved 
in  a  very  dilute  solution  of  potash  and  could  be  precipi- 
tated from  this  solution  by  the  careful  addition  of  acetic 


CHOLERA.  113 

acid,  but  dissolved  in  an  excess  of  this  acid.  It  dissolved 
also  in  a  seven  per  cent,  salt  solution,  but  was  precipi- 
tated by  a  saturated  solution.  It  g-ave  the  biuret  and 
xanthoprotein  reaction.  This  substance  proved  to  be  very- 
poisonous.  It  killed  guinea-pigs  within  twenty  minutes 
when  a  few  cubic  centimetres  of  the  alkaline  solution — 
potash — was  injected  into  the  cavity  of  the  abdomen. 
SchoU  calls  this  substance  cholera-toxo-globulin.  The 
precipitate  which  fell  to  the  bottom  of  the  receptacle  was 
washed  with  alcohol,  then  digested  with  water  for  twenty 
minutes  at  40°  C.  Very  little  was  apparently  dissolved 
out  by  this  procedure,  but  this  little  proved  to  be  very 
toxic.  In  from  one  to  three  minutes  after  the  injection 
of  a  few  cubic  centimetres  of  the  solution  into  the  peri- 
toneal cavity  of  a  guinea-pig  the  animal  died.  This 
aqueous  solution  gave  the  biuret  and  xanthoprotein  re- 
action ;  it  was  precipitated  by  mercuric  chloride,  nitrate 
of  mercury,  and  tannin,  but  not  by  a  saturated  solution  of 
ammonium  sulphate  or  acetic  acid.  This  substance 
Scholl  calls  cholera-toxo-pepton.  The  toxic  action  of 
these  substances  is  destroyed  by  a  temperature  of  100° 
C,  maintained  for  half  an  hour,  or  by  40°  to  45°  C,  main- 
tained for  twenty -four  hours.  But  at  ordinary  tempera- 
tures they  retain  their  toxic  action  for  several  weeks. 

Gruber  (1892)  has  also  obtained  a  toxic  albuminous 
precipitate  by  allowing  egg  cultures  to  fall  into  alcohol, 
drying  the  precipitate,  and  then  extracting  it  with  water. 

Gamaleia  (1893)  has  obtained  a  toxin  which  produces 
the  typical  phenomena  of  cholera,  which,  according  to 
him,  is  closely  associated  with  the  bacterial  cells,  but  can 
be  extracted  by  a  soda  solution  or  by  heating  to  55°  to 
60°  C.  The  conclusion  is  reached  that  it  is  a  nucleo- 
albumin  analogous  to  the  toxalbumins  of  tetanus  and  of 
8 


114  IMMUNITY    AND   SERUM-TIIERAPY. 

diplitlieria.  It  is  precipitated  by  alcohol,  acids,  and  by 
magnesium  sulphate. 

Fiuall}^  AVestbrook,  in  a  still  more  recent  research 
(1894),  arrives  at  the  conclusion  that  the  cholera  spirillum 
produces  various  toxic  proteids  which  in  small  amounts 
produce  immunity  in  susceptible  animals,  and  the  pro- 
duction of  which  depends  to  a  certain  extent  upon  the 
culture  medium ;  or  that  its  toxin  is  a  substance  of  con- 
stant chemical  composition  which  is  mixed  with  various 
albuminous  substances,  either  contained  in  the  culture 
medium  or  developed  in  the  culture.  Duclaux  is  of  the 
opinion  that  the  last  supposition  is  correct,  and  that  the 
so-called  toxalbumins  are  not  bodies  of  definite  chemical 
composition,  but  mixtures  of  toxins  and  albuminous  sub- 
stances. 

Experiments  made  upon  the  lower  animals  show  that 
the  introduction  of  these  cholera  toxins  into  the  bodj'  of 
a  susceptible  animal,  either  with  or  without  the  living 
cholera  spirillum,  results  in  the  establishing-  of  a  certain 
degree  of  immunity  against  the  toxic  action  of  cholera 
cultures.  And  there  is  good  reason  to  believe  that  a  non- 
fatal attack  of  cholera  in  man  gives  the  individual  a  rela- 
tive immunity  from  subsequent  attacks,  for  some  time  at 
least.  This  has  led  to  extended  experiments  with  refer- 
ence to  the  possibility  of  producing  a  similar  immunity 
in  man  by  means  of  protective  inoculations.  The  experi- 
ments bearing  upon  this  point  which  have  been  made 
uiDon  the  lower  animals  will  first  engage  our  attention. 

Huejope  (1887)  first  demonstrated  the  fact  that  injec- 
tions of  a  small  amount  of  a  cholera  culture  into  the  peri- 
toneal cavity  of  a  guinea-pig  is  fatal  to  these  animals. 

In  the  following  year  (1888)  Gamaleia  reported  his  suc- 
cess in  infecting  guinea-pigs  by  subcutaneous  injections 


CHOLERA.  115 

of  blood  from  an  infected  pigeon.  He  found  that  by  suc- 
cessive inoculations  in  pigeons  a  considerable  increase  in 
virulence  is  established,  and,  that  while  guinea-pigs  were 
not  fatally  infected  by  subcutaneous  inoculations  with 
ordinary  cultures,  they  invariably  died  when  inoculated 
with  the  more  virulent  culture  in  the  blood  of  an  infected 
pigeon.  Also,  that  when  guinea-i^igs  were  inoculated 
with  ordinary  cultures,  or  with  cultures  sterilized  by 
heat,  they  were  subsequently  immune,  and  resisted  inoc- 
ulations with  the  most  virulent  material.  In  the  same 
year  the  author  referred  to  announced  the  discovery  of  a 
sj)irillum  which  closely  resembles  the  cholera  spirillum — 
his  "  Vibrio  Metchnikovi."  This  was  obtained  from  the 
intestinal  contents  of  fowls  suffering  from  a  fatal  in- 
fectious malady  (in  Odessa).  According  to  Gamaleia 
chickens  and  pigeons  which  have  survived  an  inoculation 
with  a  culture  of  this  spirillum  are  subsequently  immune 
against  the  pathogenic  action  of  the  cholera  spirillum,  and 
vice  versa.  In  subsequent  communications  Gamaleia  re- 
ported that  sterilized  cultures  of  his  "  Vibrio  Metchni- 
kovi "  (sterilized  by  heat  at  120°  C)  were  very  pathogenic 
for  rabbits,  fowls,  pigeons,  and  even  for  dogs  and  sheep. 
The  rabl)it  proved  to  be  the  most  susceptible  animal,  and 
succumbed  to  doses  of  4  c.c.  in  from  twelve  to  twenty 
hours.  Doses  of  1  c.c.  per  100  gm.  of  body-weight 
caused  a  temporary  indisposition  followed  by  immunity. 
Pigeons  were  made  immune  by  larger  doses. 

The  researches  of  Pfeiffer  (1889)  confirmed  those  of  Ga- 
maleia as  to  the  fact  that  pigeons  and  guinea-pigs  could 
be  made  immune  against  Vibrio  Metchnikovi  by  the  injec- 
tion of  sterilized  cultures.  But  guinea-pigs  which  had 
been  immunized  against  this  pathogenic  spirillum  suc- 
cumbed to  cholera  infection  ;  and,  on  the  other  hand,  ani- 


116  IMMUNITY   AND   SERUM-THEKAPY. 

mals  which  had  been  treated,  in  various  ways  with  a  chol- 
era culture  died  without  exception  when  infected  with 
Vibrio  Metchnikovi.  The  conclusion  is  therefore  reached 
that  the  two  pathogenic  spirilla  are  distinct  species,  al- 
thoug"h  very  similar  in  many  respects. 

Brieger  and  Wassermann  (1892)  have  reported  the  re- 
sults of  experiments  with  the  cholera  spirillum  cultivated 
in  thymus  bouillon.  After  twenty -four  hours  develop- 
ment in  this  medium  the  cultures  were  sterilized  by  heat 
(55°  C.  for  fifteen  minutes)  and  placed  in  an  ice-chest  for 
twenty -four  hours.  Four  c.c.  of  this  fluid  injected  daily 
for  four  days  into  the  peritoneal  cavity  of  a  guinea-pig 
made  it  immune  to  the  cholera  spirillum  in  doses  three 
times  as  large  as  were  required  to  kill  an  animal  not  so 
treated.  This  immunity  lasted  for  two  months.  Fedoroff 
(1892)  obtained  similar  results  by  the  subcutaneous  injec- 
tion of  sterilized  cultures  in  doses  of  1  c.c,  in  guinea-i^igs. 
His  cultures  in  thymus  bouillon  were  kept  for  from  seven 
to  ten  days  at  37°  C,  then  sterilized  by  heating  for  fifteen 
minutes  at  65°  C,  then  allowed  to  stand  in  a  dark  room 
for  twenty -four  hours,  and  finally  mixed  with  an  equal 
volume  of  glycerine. 

Ketscher  (1892)  has  obtained  evidence  that  the  immu- 
nizing substance  in  animals  which  have  received  protec- 
tive inoculations  is  contained  in  the  milk  of  females  thus 
treated.  Three  goats  received  subcutaneous  inoculations 
of  virulent  cholera  cultures,  and  also  injections  into  a 
vein  and  into  the  peritoneal  cavity.  The  milk  of  these 
goats  was  injected  into  the  peritoneal  cavity  of  rabbits  ; 
these  proved  to  be  immune  when  subsequently  lethal 
doses  of  a  virulent  cholera  culture  were  injected  into 
the  peritoneal  cavity. 

According  to  Gamaleia  (1892),  dogs  are  ver}^  suscepti- 


CHOLERA.  117 

ble  to  infection  with  cholera  spirilla,  and  present  symp- 
toms closely  resembling  those  of  cholera  in  man.  They 
may  also  be  easily  immunized  against  the  pathogenic 
action  of  cholera  cultures. 

Gruber  and  Wiener  (1892)  have  also  found  that  suscep- 
tible animals  are  easily  immunized  against  cholera  infec- 
tion either  by  inoculation  with  small  doses,  with  attenu- 
ated cultures,  or  with  larger  quantities  of  sterilized 
cultures.  Haffkine  (1892)  also  reports  his  success  in  im- 
munizing guinea-pigs  and  pigeons. 

Pawlowsky  (1893)  claims  to  have  obtained  from  the 
blood  of  animals  having  an  acquired  immunity  against 
cholera  an  antitoxin  in  the  form  of  an  amorphous  pow- 
der ;  and  Lazarus  (1892)  reports  that  the  blood  of  man, 
after  recovery  from  an  attack  of  cholera,  has  the  property 
of  protecting  guinea-pigs  from  fatal  infection  when  in- 
jected, in  very  small  amount,  into  the  peritoneal  cavity. 
Issaeff  (1894)  in  an  extended  series  of  experiments  was 
not  able  to  entirely  confirm  the  results  reported  by  Laza- 
rus. In  a  summary  of  results  obtained  in  his  own  experi- 
ments he  says : 

"  1.  The  intraperitoneal  or  subcutaneous  injection  of 
blood-serum  from  normal  individuals  [that  is,  persons 
who  have  not  suffered  an  attack  of  cholera],  and  also 
of  various  acids,  alkalies,  and  neutral  liquids,  gives  to 
guinea-pigs  a  certain  resistance  against  intraperitoneal 
cholera  infection.  This  resistance,  however,  is  feeble  and 
temporary,  and  cannot  be  considered  as  identical  with 
the  true  immunity  which  results  from  vaccination  with 
the  products  of  the  cholera  bacteria. 

"  2.  Guinea-pigs  vaccinated  against  cholera  have  no 
immunity  against  the  toxins  of  the  cholera  vibrio,  not- 
withstanding their  high  degree  of  insusceptibility  to  in- 
fection with  cultures  containing  the  living  vibrio.     The 


118  IMMUNITY   AND   SERUM-THEKAPY. 

blood  of  immunized  guinea-pigs  does  not  possess  anti- 
toxic i:>ropeities.  The  maximum  dose  of  cholera  toxins 
Avhich  immune  guinea-pigs  can  withstand  is  not  greater 
than  that  which  control  animals  withstand. 

"3.  The  blood  of  guinea-pigs  carefully  immunized 
against  cholera  possesses  si^ecific  and  very  pronounced 
immunizing,  and,  in  a  certain  sense,  curative  powers. 

"4.  The  blood  of  cholera  convalescents  possesses  simi- 
lar specific  and  curative  x>owers.  This  property  is  first 
developed  about  the  end  of  the  third  week  after  the  at- 
tack, and  disappears  completely  at  the  end  of  two  or 
three  months." 

In  a  series  of  experiments  made  by  Pfeiffer  and  Issaeff 
the  results  obtained,  as  stated  by  Pfeiffer  in  a  subsequent 
communication,  were  as  follows  : 

"  In  my  research  with  Issaeff  '  uj)on  the  explanation  of 
cholera  immunity '  I  proved  that  the  serum  of  animals 
which  have  an  active  acquired  immunity  against  cholera 
only  has  a  specific  action  upon  this  particular  species  of 
vibrio,  and  as  regards  other  species  of  bacteria  does  not 
difier  in  its  action  from  the  blood-serum  of  normal  ani- 
mals. We  also  showed  that  this  specific  influence  in  re- 
spect to  the  intraperitoneal  cholera-infection  of  guinea- 
pigs  was  due  exclusively  to  bactericidal  processes  which 
in  some  way  were  induced  by  the  serum  of  immune  ani- 
mals." 

The  view  of  Pfeiffer,  founded  upon  his  experimental 
results,  is  that  the  destruction  of  the  living  cholera  spi- 
rilla, which  quickly  takes  place  "in  the  peritoneal  cavity  of 
the  guinea-pig,  when  at  the  same  time  a  minute  quantity 
of  serum  from  an  immune  animal  is  introduced,  is  not 
directly  due  to  the  bactericidal  action  of  this  serum,  but 
that,  in  some  way,  it  gives  rise  to  a  specific  bactericidal 
action  in  the  exudate  which  is  found  in  the  peritoneal 


CHOLERA.  119 

cavity  as  a  result  of  such  injections.  His  experiments 
also  lead  liiin  to  the  conclusion  that  this  is  accomplished 
quite  independently  of  phagocytosis. 

The  brief  review  of  experimental  researches  relating  to 
cholera  immunity  which  we  have  made  shows  that,  while 
there  is  a  general  agreement  as  to  the  possibility  of  pro- 
ducing immunity  in  susceptible  animals,  there  is  consid- 
erable diiference  of  opinion  as  to  the  true  explanation  of 
this  immunity.  The  supposition  that  it  is  due  to  an  an- 
titoxin which  has  the  power  of  neutralizing  the  toxic 
products  of  the  cholera  spirillum  does  not  receive  any 
support  from  the  most  recent  investigations — those  of 
Pfeiflfer  and  Issaeff — which,  on  the  contrary,  seem  to  es- 
tablish the  fact  that  this  immunity  depends  upon  an  in- 
creased bactericidal  activity  of  the  blood-serum  of  im- 
mune animals,  A  very  curious  fact  developed  by  the 
researches  of  the  bacteriologists  last  named  is  that— 

"  The  cholera-serum,  which  in  the  peritoneal  cavity  of 
guinea-pigs  acted  only  upon  the  cholera  bacteria,  and 
behaved  toAvard  other  vibrios  exactly  like  the  serum  of 
normal  animals,  in  a  test-tube  killed  all  four  species  of 
vibrios  with  equal  rapidity." 

Unfortunately  the  evidence  relating  to  the  value  of  pro- 
tective inoculations  in  man,  although  supported  by  the 
evidence  already  referred  to  as  regards  the  lower  ani- 
mals, is,  to  a  considerable  extent,  unsatisfactory,  owing 
to  the  difficulty  of  applying  scientific  methods  to  experi- 
ments of  this  kind.  The  evidence,  however,  is  in  favor 
of  the  view  that  a  certain  degree  of  protection  is  afforded 
by  the  subcutaneous  injection  of  cholera  cultures.  Such 
protective  inoculations  could  not  be  expected  to  confer 
an  absolute  immunity,  inasmuch  as  the  immunity  result- 


120  IMMUNITY   AND   SERUM-THERAPY. 

ing  from  a  single  attack  has  only  a  relative  value,  and  is 
probably  not  of  long  duration. 

We  quote  from  Shakespeare's  "  Rei)ort  on  Cholera  in 
Europe  and  India,  1890,"  the  following  jaaragraphs  re- 
lating to  immunity  as  a  result  of  an  attack  of  cholera : 

"immunity    after  an    attack    of    cholera — EXPERIENCE  IN 
FRANCE,  1884. 

"  The  Academy  of  Medicine  of  Paris  directed  a  circular 
letter  of  questions  concerning  cholera  to  the  physicians 
of  the  localities  infected  by  that  disease  in  1884,  and  in 
group  L  of  general  observations  in  that  questonario  is 
found  the  following :  '  Have  there  been  observed  recur- 
rences among  the  peojDle  attacked,  either  in  a  former  epi- 
demic or  in  the  present  one  ?  Give  the  results  of  this  re- 
currence.' In  response  to  their  questions  the  Academy 
received  184  communications,  but  the  committee  ap- 
pointed to  analyze  them  eliminated  79 ;  for  various  rea- 
sons given  only  104  were  used  for  analysis.  Of  this 
number  only  8  bore  upon  the  i:)articular  question  above 
mentioned,  and  it  is  reasonable  to  assume  that  the  other 
96  observers  said  nothing  concerning  this  point  because 
they  had  observed  nothing  bearing  upon  it.  The  results 
of  this  analysis  may  be  stated  as  follows  : 

"  From  Castelnaudary,  with  a  population  of  10,000,  we 
learn  that  there  were  54  cases  and  18  deaths  from  cholera, 
among  which  there  was  1  recurrence  ;  from  Aix,  with  20,- 
257,  number  of  cases  unknown,  deaths  117,  among  these 
2  recurrences  were  observed,  at  intervals  of  ten  and  forty 
days  ;  from  Beseges,  with  11,400  inhabitants,  we  learn  of 
124  cases  and  40  deaths,  among  which  were  2  recurrences  ; 
from  Cette,  with  35,000,  the  number  of  cases  is  not  men- 
tioned, but  we  learn  that  there  were  92  deaths  and  1  re- 
currence ;  from  Nantes,  with  124,300  inhabitants,  we  learn 
of  251  cases  and  112  deaths,  vvitli  1  recurrence  ;  from  Per- 
pignan,  with  25,000  inhabitants,  we  hear  of  325  cases  and 
225   deaths,   and  receive   the .  indefinite   statement   that 


CHOLEEA.  121 

there  were  some  fatal  recurrences  ;  from  Pignans,  popu- 
lation not  stated,  we  learn  of  22  attacks  and  12  deaths, 
with  1  recurrence ;  from  Cadenet,  with  a  poiDulation  of 
26,000,  we  are  not  informed  of  the  number  of  cases,  but 
learned  that  there  were  20  deaths  and  2  recurrences." 

"  IMMUNITY    AFTER  AN    ATTACK    OF    CHOLERA — EXPERIENCE   IN 

SPAIN,  1885. 

"While  examining-  cholera  in  Spain,  the  writer  pre- 
pared a  circular  containing-  a  series  of  twenty-live  ques- 
tions relating  especially  to  the  nature,  etiology,  and  proph- 
ylaxis of  cholera,  one  of  which  requested  the  physician 
to  state  whether  or  not,  in  his  own  personal  experience, 
he  had  observed  a  second  or  a  third  attack  of  cholera  dur- 
ing the  same  epidemic,  and  in  case  of  a  positive  reply  to 
detail  the  symptoms  and  all  the  circumstances  surround- 
ing it.  This  circular  -  letter  was  addressed  to  some 
twenty-five  hundred  Spanish  physicians,  located  in  the 
various  cities,  towns,  and  villages  in  that  kingdom  which 
had  suffered  from  the  eiDidemic.  Among  the  large  num- 
ber of  replies  there  were  only  8  in  which  a  second  attack 
was  reported,  and  from  an  examination  of  the  details  of 
these  there  was  no  doubt  left  in  our  mind  that  6  were 
not  genuine  second  attacks  after  a  complete  recovery, 
but  were  in  reality  relapses  due  to  imprudences  of  diet  or 
otherwise  before  convalescence  and  complete  recovery 
had  been  established.  Two  of  the  8  cases,  from  the 
details  of  the  reports  given,  may  have  been  genuine  re- 
current attacks  of  Asiatic  cholera,  or  may  have  been  sim- 
ply seizures  of  cholera  morbus  (cholera  nostras).  It  is 
well  known  that  after  an  attack  of  Asiatic  cholera  the 
digestive  apparatus  is  left  in  a  damaged  condition,  and 
disorders  of  the  intestines  continue  for  a  long  time.  The 
habits  of  life  and  the  imprudences  so  common  to  the  class 
of  people  most  frequently  suffering  from  Asiatic  cholera 
in  that  country  are  such  as  to  render  them  more  than  usu- 
ally liable  to  suffer  attacks  of  cholera  nostras.  As  having 
an  important  bearing  upon  this  suggestion,  the  writer 


122  IMMUNITY   AND   SERUM-TIIEHAPY. 

made  an  analysis  of  tlie  vital  statistics  of  Spain,  covering 
the  five  years  previous  to  1885,  for  the  purpose  of  learn- 
ing the  extent  of  prevalence  of  cholera  nostras  among 
that  population,  and  the  result  of  the  inquiry  shows  that 
the  number  of  deaths  attributed  to  that  disease  averaged 
per  year  sixteen  per  every  million  inhabitants." 

Dr.  Ferran,  who  practised  inoculations  on  an  extensive 
scale  during  the  epidemic  of  1885,  in  Spain,  gives  the  fol- 
lowing account  of  his  method  of  performing  these  inocu- 
lations : 

"  1.  The  cholera  vaccine  is  nothing  more  than  a  pure 
culture,  in  bouillon,  of  the  comma  bacillus.  Its  easy  and 
long  preservation  (four  to  five  days)  allows  of  its  trans- 
portability to  great  distances,  taking  care  always  to  keep 
the  flask  which  contains  the  material  upright. 

"  2.  Heat  and  cold  do  not  interfere  with  its  preserva- 
tion if  the  vaccine  is  to  be  used  in  a  short  time.  It  should 
not,  however,  be  kept  out  of  doors  during  the  warm  sea- 
son. 

"  3.  The  vaccine  should  be  kept  in  flasks  of  the  model 
of  Ferran,  with  a  flat  bottom  and  a  short  neck.  The  stop- 
per, which  is  of  rubber,  fits  perfectly,  and  is  penetrated 
by  two  glass  tubes,  one  straight  and  short,  which  does 
not  extend  below  the  inferior  surface  of  the  stopper,  and 
which  does  not  project  above  more  than  some  two  centi- 
metres, is  plugged  with  a  small  quantity  of  sterilized  cot- 
ton and  a  superficial  covering  of  wax.  The  other  glass 
tube  is  longer,  and  extends  on  the  lower  side  as  far  as 
the  bottom  of  the  flask,  while  its  superior  end  is  curved, 
and  terminates  in  a  capillary  extremity,  the  tip  of  which 
is  closed  with  wax. 

"  4.  When  the  vaccine  is  to  be  used  it  is  necessary  to 
make  two  principal  preparations  for  the  operation.  A 
small  syringe  for  the  hypodermic  injection,  and  a  small 
vessel  into  which  it  is  necessary  to  empty  the  fluid  from 
the  flask  are  required.     The  syringe  should  have  me- 


CHOLERA.  123 

tallic  pistons  and  mountings,  witlioiit  mastic  of  any  kind 
and  without  rubber.  Its  capacity  should  be  1  c.c,  its 
needle  thicker  and  shorter  than  that  of  ordinary  use. 
Before  beginning  the  vaccination  the  syringe  must  be 
tilled  two  or  three  times  with  boiling  water,  which  is 
aspirated  and  expelled  through  the  needle.  This  is  called 
sterilizing  the  instrument,  and  by  this  means  the  ex- 
traneous germs  are  destroyed  which  might  be  contained 
in  it,  in  order  to  avoid  the  production  of  phlegmons 
and  abscesses.  The  trouble  in  taking  this  precaution 
will  be  little.  Acting  thus,  one  may  perform  thousands 
of  injections  without  fear  of  any  accident.  It  is  sug- 
gested that  it  is  a  bad  custom  to  pass  the  needle  through 
a  Hame  in  order  to  sterilize  it,  because  this  mode  of  pro- 
cedure draws  the  temper.  Another  precaution  that  must 
be  taken  relates  to  the  examination  of  the  syringe  before 
using  it,  in  order  to  be  well  assured  that  the  piston  acts 
Ijerfectly  and  that  not  a  single  drop  of  the  liquid  escapes 
by  a  leak  in  the  cannula.  This  latter  defect  is  sufficient  to 
reject  the  instrument.  If  the  syringe  aspires  air  because 
the  leather  washer,  which  is  placed  at  the  end  of  the  glass 
tube  in  order  to  facilitate  its  adaptation  is  dry,  or  the  pis- 
ton is  in  the  same  condition,  it  is  necessary  to  dela^^  a 
little  while  in  order  to  take  the  syringe  apart  and  soak  it 
in  warm  water.  It  is  convenient  to  keep  several  syringes 
for  use,  with  a  sufficient  number  of  needles,  when  many 
inoculations  are  to  be  performed. 

"  5.  The  small  receptacle  into  which  the  vaccine  is 
poured  in  order  that  the  syringe  may  be  filled  readily  is 
a  capsule,  a  cup,  or  some  similar  vessel.  Before  use,  it 
should  be  washed  and  dried  with  extreme  care,  and  im- 
mediately before  using  passed  through  an  alcohol  or 
Bunsen  flame,  in  order  to  sterilize  it. 

"  6.  All  these  preparations  having  been  made,  the 
drop  of  wax  which  closes  the  capillary  extremity  of  the 
long  tube  of  the  flask  is  removed,  and  at  the  same  time 
also  the  wax  covering  of  the  cotton-stopper  of  the  short 
tube,  but  by  no  means  must  this  cotton  stopper  be  re- 


124  IMMUNITY   AND   SERUM-THERAPY. 

moved  ;  a  rubber  tube,  or  the  extremity  of  a  small  Rich- 
ardson spray  apparatvis,  is  adjusted  to  the  short  tube. 
The  capillary  extremity  of  the  loug-  tube  is  now  slightly 
warmed  in  order  to  soften  somewhat  the  wax  which  may 
have  been  drawn  into  its  lumen  by  capillarity,  and  air  is 
forced  into  the  flask,  either  by  blowing-  into  the  rubber- 
tube  or  by  working-  the  Richardson  atomizer  ;  the  air  in- 
jected by  pressure  upon  the  vaccine  fluid  forces  the  latter 
out  through  the  long  tube  witli  the  capillary  extremity, 
and  it  is  collected  in  the  cup  or  small  sterilized  vessel. 
This  latter  is  then  covered  with  white  paper,  which  has 
been  scorched  in  the  flame,  or  with  a  sterilized  glass 
plate  ;  as  often  as  the  syringe  is  filled,  this  cover  will  be 
removed  and  again  immediately  afterward  replaced. 

"  7.  Never  should  the  rubber  stopper  which  closes  the 
flask,  or  the  cotton  which  plugs  the  short  straight  tube,  be 
removed,  because  otherwise  the  germs  of  the  external  air 
might  enter  and  contaminate  the  culture,  and  in  this  way 
give  place  to  local  and  general  accidents  among  the  inoc- 
ulated. Whenever,  through  the  movements  of  transpor- 
tation, the  cotton  plug  in  the  short  glass  tube  has  be- 
come so  wet  as  to  impede  the  passage  of  the  air  which  is 
to  be  forced  into  the  flask  in  the  act  of  expelling  the  vac- 
cine from  it,  it  may  be  removed  with  the  point  of  a  needle 
and  rapidly  substituted  by  another  plug  of  svirgical  cot- 
ton which  has  been  carbonized  or  salicylized.  If  this 
proceeds  with  cleanness  and  promptness,  there  is  no  dan- 
ger in  doing  it.  When  the  cotton,  although  wet,  does 
not  impede  the  injection  of  the  air,  it  is  better  not  to 
change  it. 

"  8.  After  terminating  the  vaccination,  again  the  cap- 
illary extremity  of  the  curved  tube  is  passed  through  the 
flame  until  the  small  quantity  of  liquid  remaining  in  it  is 
evaporated  ;  it  is  then  stopped  a  second  time  with  a 
small  drop  of  wax  ;  and  from  the  other  glass  tube  the 
rubber  tube  which  has  been  employed  for  forcing  in  the 
air  is  removed  and  another  thin  layer  of  wax  is  placed 
over  the  cotton  plug. 


CHOLERA.  125 

"9.  If  in  the  smaller  vessel  or  cup  any  of  the  vaccine 
fluid  remains  after  the  vaccination  of  all  persons  present, 
it  is  boiled,  and  in  this  manner  the  culture  is  killed,  for 
it  should  not  be  used  in  another  operation,  because  at- 
mospheric germs  mig-ht  become  mixed  with  it. 

"  10.  The  technique  for  the  iDractice  of  the  inoculation 
is  the  same  as  for  all  hyi^odermic  injections.  The  most 
convenient  region  is  that  of  the  brachial  triceps. 

"  11.  The  dose  is  1  c.c. — or  the  contents  of  a  syringe — 
into  each  arm,  for  individuals  of  all  ages  and  conditions. 

"  12.  Five  days  having  elapsed,  revaccination  may  be 
performed  by  following  the  same  instructions." 

Shakespeare,  who  was  sent  by  the  United  States  Gov- 
ernment to  Spain  to  investigate  the  results  of  these  in- 
oculations, reports  as  follows  : 

"  And  now  with  respect  to  the  human  inoculations : 
The  most  of  these  inoculations  were  performed  in  villages 
in  the  province  of  Valencia.  The  number  of  persons  in- 
oculated considerably  exceeds  thirty  thousand.  Much  has 
been  both  said  and  written  in  Spain,  France,  and  Eng- 
land concerning  the  results  of  these  inoculations.  The 
results  which  have  been  published  have  appeared  to  very 
strongly  back  up  the  claim  of  Dr.  Ferrdn  that  choleraic 
inoculation  has  the  power  of  protecting  the  individual 
against  an  attack  of  cholera,  and  that  the  extensive  prac- 
tice of  this  inoculation  among  villages  already  invaded 
by  the  epidemic  is  a  powerful  and  at  the  same  time  harm- 
less means  of  bringing  the  epidemic  to  an  end.  This 
being  the  case,  for  those  who  were  unwilling  to  accept 
the  deductions  to  be  made  from  the  published  statistics, 
the  only  way  of  escaping  their  force  seemed  to  be  by  an 
attack  upon  their  validity. 

"  The  statistics  of  the  anti-choleraic  inoculations  have 
been  widely  attacked.  The  first  public  onslaught  upon 
these  statistics  of  which  the  world,  outside  of  Spain,  had 
much  knowledge,  was  made  in  the  report  of  the  French 


126  IMMUNITY    AND   SEEUM-THEEAPY. 

Commissiori,  with  Dr.  Brouardel  at  its  head,  which  "vvas 
presented  to  the  Minister  of  Commerce  after  the  return  of 
that  Commission  from  Spain  in  the  summer  of  1885.  It 
is  charged  in  that  report  that  the  results  of  the  statistics 
therein  reproduced  are  assailable  on  account  of  having- 
been  collected  by  physicians  Avho  were  partisan  support- 
ers of  Dr.  Ferran,  and  that  they  neither  possessed  any 
adequate  official  character,  nor  did  they  possess  suffi- 
cient details.  As  far  as  I  can  learn,  the  general  impres- 
sion entertained  throughout  the  world  of  the  value  of  the 
inoculation  statistics  is  based,  in  the  main,  upon  this  re- 
IDort  of  the  French  Commission. 

"  The  statement  of  that  Commission  that  the  statistics 
which  they  had  been  able  to  obtain  of  the  preventive  in- 
oculations of  Ferran  were  to  a  considerable  degree  void 
of  any  official  character  may  be  true,  and  perhaps  it  is 
also  true  that  they  emanated  from  the  jDartisan  friends  of 
Ferran;  but  it  must  be  distinctly  remembered  that  at 
that  day  there  were  practically  no  official  statistics  of  this 
kind  in  the  hands  of  anyone.  The  official  statistics  col- 
lected under  the  orders  of  the  Spanish  Government  were 
gotten  together  at  a  far  later  date. 

"  Ui^on  the  appointment  of  the  Government  at  Madrid 
of  the  second  official  Spanish  Commission  to  investigate 
the  Ferran  question  in  the  provinces  where  the  inocula- 
tions were  being  practised,  it  was  ordered  that  official 
statistics  of  the  inoculations  should  be  collected  in  the 
usual  manner  ;  that  is  to  say,  by  the  customary  statistical 
officers  of  the  Government.  This  second  medical  Com- 
mission was  also  accompanied  by  an  independent  statisti- 
cal commission  who  were  charged  with  the  duty  of  form- 
ing statistics  of  those  inoculations  which  were  expected 
to  be  witnessed  by  the  Medical  Commission  in  their  tour 
of  investigation,  and  the  report  to  the  Spanish  Govern- 
ment of  this  statistical  commission  is  based  exclusively 
upon  the  official  statistics  which  thej^  themselves  col- 
lected. 

"  In  estimating  the  value  of  the  official  character  and 


CHOLERA.  127 

the  authority  of  the  official  statistics,  which  have  since  the 
visit  of  the  F'rencJi  Conuidssioa  to  Spain  been  collected  aud 
published,  the  following-  circumstances  should  be  taken 
into  account :  The  provincial  governments  of  Spain  are 
somewhat  peculiar,  in  that  the  civil  governors  change 
with  the  changes  which  take  place  in  the  Government  at 
Madrid,  so  that  the  political  constitution  of  the  provin- 
cial governments  is  always  a  reflex  of  that  of  the  central 
Government  at  Madrid.  Moreover,  the  i:)olitical  senti- 
ment of  the  provincial  government  is  also  more  or  less 
perfectly  reflected  by  the  local  governments  of  the  towns 
of  the  province. 

"  The  hostility  of  the  Minister  of  the  Interior  at  Ma- 
drid to  Dr.  Ferran,  and  his  attempts  at  the  prevention  of 
cholera  by  inoculation,  is  a  well-known  fact  now  gener- 
ally admitted  ;  and  the  hostility  which  Dr.  Ferran  met 
with  from  the  civil  governor  of  the  province  of  Valencia 
was  even  greater  than  that  manifested  by  the  Minister  of 
the  Interior  himself. 

"  The  official  statistics  of  the  Ferran  inoculations  are 
in  the  first  place  signed  by  the  physicians  of  the  locality ; 
and  in  the  next  place  by  the  judge  of  the  municipal  court, 
and  sometimes  also  by  the  president  judge  of  the  judicial 
district,  by  the  parochial  priest,  and  by  the  mayor  of  the 
municipality,  whose  signatures  and  seals  are  attested  by 
an  authorized  notary  public. 

"  It  must,  therefore,  be  obvious  that  the  charge  made 
by  the  French  Commission,  which  has  been  so  constantly 
reiterated  everywhere,  that  the  public  statistics  of  the 
anti-choleraic  inoculations  are  void  of  official  character 
and  are  to  be  regarded  as  ex-parte  testimony  of  the  j)arti- 
sans  of  Ferran,  cannot  apply  to  official  statistics  which 
were  collected  under  the  supervision  of  the  municijial 
authorities  of  the  villages  wherein  the  inoculations  were 
performed,  and  attested  not  only  by  the  local  judicial 
officers  and  the  parochial  priests,  but  also  by  the  political 
officers — that  is  to  say,  the  secretaries  and  the  mayors  of 
the  municipalities  ;  for  it  must  be  admitted  that  neither 


128  IMMUNITY   AND   SERUM-THERAPY. 

the  political  officers  of  the  municipalities  nor  of  the  pro- 
vincial governments,  any  more  than  the  parochial  priests, 
can  reasonably  be  charged  with  being  the  partisans  or 
friends  of  Ferran — the  Minister  of  the  Interior  continuing 
during  the  time  of  collection  of  these  official  statistics  to 
be  hostile  to  the  claims  of  Ferrd,n.  It  therefore  follows 
that  the  attack  upon  the  statistics  of  the  inoculations 
made  by  the  French  Commission,  and  so  widely  accepted 
by  the  medical  world  as  conclusive,  does  not  apply  to  the 
official  statistics  of  which  we  are  speaking.  And,  in  view 
of  this  fact,  the  evidence  as  to  the  efficiency  and  harm- 
lessness  of  the  anti-choleraic  inoculations  should  be  re- 
examined. As  I  have  already  said,  the  results  of  the 
preventive  inoculations  of  Ferran,  as  set  forth  in  the  offi- 
cial statistics,  appear  to  verj^  strongly  support  his  claim 
of  the  protective  value  of  the  inoculations.  In  view  of 
the  great  importance  of  this  whole  subject  I  have  deter- 
mined to  place  these  statistics  in  this  report  for  the  bene- 
fit of  the  readers  of  the  English  language,  in  order  that 
they  may  judge  for  themselves  of  the  facts  as  they  appear 
to  be  recorded. 

■"  From  the  Government  statistics  of  cholera  throughout 
the  province  of  Valencia,  it  appears  that  among  the  vil- 
lages invaded  there  were  62  attacks  per  thousand  of  the 
population,  and  31  deaths  per  thousand,  which  gives  a 
mortality  of  fifty  per  cent,  of  those  attacked.  It  appears 
from  analysis  of  the  published  official  statistics  of  cholera 
in  twenty-two  towns  where  inoculation  Avas  performed 
the  inhabitants  were  divided  as  follows  :  104,561  not  in- 
oculated ;  30,491  inoculated.  Of  the  latter  there  were 
387  attacks  of  cholera,  or  12  per  thousand,  and  104  deaths, 
or  8  per  thousand  ;  the  mortality  of  those  attacked  being 
twenty-five  per  cent.  Of  the  former  there  were  8,406  at- 
tacks, or  77  per  thousand,  and  3,512  deaths,  or  33  per 
thousand,  being  a  mortality  of  those  attacked  of  forty - 
three  per  cent.  It  appears,  therefore,  that  among  the 
population  of  villages  wherein  anti-choleraic  inoculations 
had  been  more  or  less  extensively  performed  the  liabil- 


CHOLERA.  129 

ity  of  the  inoculated  to  attacks  of  cholera  was  6.06  times 
less  than  that  of  the  non-inoculated,  whilst  the  liability 
of  the  inoculated  to  death  by  cholera  was  9.87  times 
less  than  that  of  the  non-inoculated.  These  figures  are 
based  exclusively  upon  the  data  furnished  by  inocula- 
tions, the  reinoculations  being-  left  out  of  consideration, 
because  they  are  much  less  numerous,  althoug-h  from  the 
records  of  the  inoculations  it  would  seem  that  the  liabil- 
ity of  attack,  and  especially  of  death  by  cholera  is  many 
times  less  among  them  than  among  those  inoculated  a 
single  time. 

"  The  charge  has  also  been  made  with  resj^ect  to  the 
published  records  of  the  inoculations  that  the  hygienic 
and  physical  condition  of  the  subjects  of  inoculation 
have  not  been  sufficiently  indicated  in  the  records,  and 
that  the  vast  majority  of  those  profiting  by  the  oppor- 
tunity to  receive  the  anti-choleraic  inoculations  were  of 
the  middle  and  upper  classes,  and  therefore  not  of  that 
class  of  the  inhabitants  who  are  notoriously  most  liable 
to  attack  and  death  from  cholera.  This  criticism  may 
have  some  justness  as  respects  some,  perhaps  many,  of 
the  villages  where  inoculations  were  performed ;  but 
there  are  certainly  many  of  the  villages  wherein  the  re- 
sults of  the  inoculation  seemed  to  be  most  positively 
in  favor  of  the  claim  of  Ferran  where  this  criticism  can- 
not hold.  I  refer  to  villages  wherein  three-fourths  or 
four-fifths  of  the  inhabitants  were  inoculated,  leaving 
only  the  fraction  of  the  ijopulation  non-inoculated.  Even 
in  the  absence  of  any  special  notes  indicating  the  social 
conditions  and  hygienic  surroundings  of  the  inoculated 
in  these  villages  it  is  ridiculous  to  assume  that  the  vast 
majority  of  these  were  peojile  of  the  middle  and  upper 
classes,  and  were  therefore  but  little  liable  to  attack  and 
death  by  cholera.  Anj^  one  acquainted  with  the  char- 
acter of  the  Spanish  population  as  it  exists  in  the  rural 
villages,  will  admit  at  once  that  the  vast  majority  of  this 
population  consists  of  the  wretched  and  the  poor,  who 
live  under  the  most  unhygienic  and  unsalubrious   con- 


130  IMMUNITY   AND   SERUM-THERAPY. 

ditions,  and  therefore   are  of  that  class  most  liable  to 
suffer  from  cholera. 

"  There  is  still  another  result  of  the  preventive  inocu- 
lations of  Ferrau  apparently  shown  by  these  statistics. 
I  refer  to  the  apparent  marked  shortening-  of  the  course 
of  the  epidemic  after  a  large  percentag-e  of  the  inhabi- 
tants have  become  inoculated.  It  would  seem,  therefore, 
from  analysis  of  the  official  statistics,  that  the  practice  of 
the  anti-choleraic  inoculation  after  the  method  of  Ferrau, 
besides  g-iving  the  subject  inoculated  a  considerable  im- 
munitj'  from  attack  and  death  by*  cholera,  furnishes  a 
means  of  bringing  an  epidemic  rapidly  to  an  end." 

With  reference  to  Haffkine's  method  of  inoculation  we 
cannot  do  better  than  to  quote  from  a  lecture  which  he 
g-ave  in  London,  in  1893  : 

"  In  the  research  that  I  have  done  at  the  Pasteur  Insti- 
tute on  vaccination  against  Asiatic  cholera  I  have  chosen 
for  my  starting-point  the  inoculation  of  the  animal  into  the 
peritoneal  cavity.  Starting  from  this  point  I  have  worked 
out  a  method  which  permits  the  culture  of  the  microbe 
in  the  animal  organism  in  a  state  of  purity  during  indef- 
inite generations,  the  exaltation  of  it  to  a  well-deter- 
mined maximum  of  strength,  and  keeping  it  at  the  same 
degi-ee  of  virulence  for  an  unlimited  period  of  time. 

"  This  method  is  illustrated  by  three  series  of  exjjeri- 
ments  which  were  the  subject  of  our  publications  in  the 
Comptes-rendus  de  la  Societe  de  Biologie  of  Paris,  and 
which  are  : 

"  1.  Giving  the  first  animal  a  dose  larger  than  the  fatal 
dose,  and  killing  this  animal  in  a  sufficiently  short  space 
of  time  to  be  able  to  find  the  more  resisting  microbes. 

"  2.  To  expose  the  exudation  taken  from  the  peritoneal 
cavity  to  the  air"  for  several  hours. 

"  3.  Then  to  transfer  this  exudation  to  the  next  animal, 
of  large  or  small  size,  according  to  the  concentration  of 
the  exudation. 


CHOLERA.  131 

"In  the  hands  of  a  number  of  other  experimenters  this 
method  has  given  the  same  results  and  showed  a  perfect 
consistency. 

"  The  properties  of  the  virus  which  is  obtained  in  this 
manner  of  cultivation  are  as  follows  :  Upon  intraperito- 
neal inoculation  it  kills  guinea-iDigs  regularly  in  the 
space  of  about  eight  hours,  and  the  fatal  dose  for  this 
animal  is  reduced  to  about  twenty  times  less  than  that 
which  it  would  have  been  necessary  to  take  for  the  mi- 
crobe with  which  I  started.  The  same  inoculation  kills 
rabbits  and  pigeons  with  a  dose  which  Avould  have  been 
perfectly  harmless  at  the  beginning  of  the  experiments. 
It  kills  guinea-pigs  by  intramuscular  inoculation. 

"The  subcutaneous  inoculation  brings  about  the  forma- 
tion of  a  large  oedema,  which  tends  toward  sequestration 
of  a  whole  part  of  the  cutaneous  tissues  and  to  the  for- 
mation of  a  wide  open  wound,  which  is  cured  in  from 
two  to  three  weeks. 

"  The  basis  of  anticholeraic  vaccination  is  founded  on 
the  virus  obtained  in  the  manner  we  have  just  described. 

"  This  virus,  injected  under  the  skin  of  a  healthy  ani- 
mal, gives  it,  after  several  days,  immunity  from  all  chol- 
eraic contamination,  in  whatever  manner  this  may  arise ; 
that  is  to  say,  if  an  animal  that  has  been  thus  treated  be 
taken,  and  an  attempt  made  to  infect  it  either  by  the  di- 
gestive canal,  by  neutralization  of  the  gastric  juice  and 
the  injection  of  opium  into  the  peritoneum,  or  by  the  in- 
troduction of  the  microbe  into  the  intestines  by  the 
method  of  Nicati  and  Rietsch,  or  by  intramuscular  inoc- 
ulation, or  finally,  by  intraperitoneal  injection,  the  most 
terrible  of  all,  it  resists,  whilst  the  control  animals  suc- 
cumb. 

"  Anticholeraic  vaccination  of  animals  in  this  manner 
is  then  definitely  established.  But  the  operation  de- 
scribed cannot  be,  such  as  it  is,  applied  to  man.  The 
wound  following  on  the  subcutaneous  inoculation  is  ter- 
rible to  look  at,  and,  in  all  probability,  extremely  painful. 
Besides,  although  it  does  not  in  itself  present  any  danger 


132  IMMUNITY   AND   SERUM-THERAPY. 

to  the  health  of  the  individual,  it  exposes  him  to  all  the 
complications  inseparable  from  an  open  wound. 

"  This  power  of  producing  necrosis  of  the  cutaneous 
tissues  has  been  removed  from  the  exalted  vaccine  by 
cultivating-  it  at  a  temperature  of  39°  C,  and  in  an  atmos- 
phere constantly  aerated.  Under  these  conditions  the 
first  generations  of  the  cholera  microbe  would  die  rapidly, 
in  an  interval  of  two  to  three  daj's,  and  therefore  care 
must  be  taken  to  sow  them  again  in  new  media  immedi- 
ately before  death,  and  after  a  series  of  generations  of 
this  kind  a  culture  is  obtained  which,  if  injected  under 
the  skin  of  animals,  even  in  exaggerated  doses,  only  pro- 
duces a  passing  oedema,  and  prepares  the  organism  in 
such  a  manner  that  the  injection  of  exalted  virus,  the 
definite  vaccine,  only  produces  a  local  reaction  of  the 
slightest  description. 

"vaccination  by  fixed  vacceste. 

"  The  method  of  vaccination  thus  worked  out  comprises, 
then,  two  vaccines — a  mild  vaccine,  obtained  by  weaken- 
ing the  fixed  virus  ;  and  a  strengthened  vaccine,  which  is 
presented  by  the  virus  itself.  It  is  easy  to  understand 
why  to  obtain  the  weakened  vaccine  we  do  not  use  an 
ordinary  virus,  but  a  virus  the  nature  of  which  has  been 
previously  fixed  in  the  laboratory.  It  is  because  the 
virus,  such  as  is  found  in  the  natural  state,  especially 
when  it  has  a  saprophytic  phase  of  development,  presents 
such  pathogenic  difterences  that  there  is  no  certainty  in 
its  application.  Respecting  this  we  need  only  recall  the 
story  of  variolization,  and  the  great  danger  that  an  indi- 
vidual incurred  when  the  infectious  substance  from  a 
slightly  attacked  subject  was  transferred  to  him.  The 
mildness  or  the  gravity  of  an  infection  does  not  depend 
only  on  the  veritable  strength  of  the  contagious  sub- 
stance, but  upon  the  resistance  of  the  individual  fi'om 
whom  it  is  taken.  Thus  it  happened  that  in  taking 
vaccine  lymph  from  a  subject  lightly   aflected,  a  very 


CHOLERA.  133 

weak  substance  was  sometimes  produced,  whicli  was. in- 
capable of  producing'  a  protective  action  ;  and  sometimes 
a  lymph  of  suck  strength  that  it  killed  less  resistant  in- 
dividuals. The  great  benefit  of  Jenner's  discovery  lay  in 
that  it  precisely  indicated  a  substance  fixed  by  passages 
through  animals,  and  of  a  virulence  below  that  which  is 
fatal  to  the  human  organism.  Another  example  is  given 
in  the  method  of  Toussaint  of  vaccination  against  an- 
thrax, the  first  of  its  kind,  which  has  been  obliged  to 
make  way  for  the  method  of  M.  Pasteur,  for  the  sole 
reason  that  the  latter,  based  upon  virus  of  a  fixed  nature, 
presented  an  absolute  ceiiiainty  in  its  results  which  was 
wanting  in  the  other.  Finally,  in  the  history  of  cholera 
itself  I  may  recall  the  attempt  made  in  1885  by  Dr.  Fer- 
ran,  of  Barcelona,  who,  with  the  object  of  j) reserving  the 
population  of  the  Peninsula  from  the  epidemic  of  cholera, 
made  injections  in  his  patients  of  the  ordinary  virus 
taken  from  dead  bodies  and  cultivated  in  the  laboratory. 
The  statistics  of  the  results  obtained  by  this  means 
showed  such  uuceiiaintj^  that  no  one  dared  to  recommend 
this  operation  to  his  country  in  spite  of  the  very  numer- 
ous tiials  made  in  Spain. 

"  The  possibility  of  treating  the  animal  organism  by 
vaccines  of  an  absolutely  fixed  nature,  prepared  by  means 
of  special  operations,  constitutes,  on  the  contrary,  the 
basis  of  the  Pasteurian  method,  and  here  lies  the  whole 
secret  and  the  sole  g-uarantee  of  the  success  of  its  appli- 
cation. 

"  APPLICATION   or  THE    IVIETHOD  TO  MAN. 

"  The  method  of  anticholeraic  vaccination,  worked  out 
by  experiments  on  guinea-pigs,  was  tried  upon  rabbits 
and  pigeons  before  it  was  applied  to  man.  These  ani- 
mals were  chosen  in  order  to  have  subjects  very  difi^er- 
ently  organized,  and  in  order  to  be  able  to  generalize  the 
conclusions,  and  to  be  able  to  extend  them  to  the  human 
organism. 

*'  The  result  obtained  on  all  these  animals  being  abso- 


134  IMMUNITY   AND   SERUM-THERAPY. 

lutely  the  same,  it  was  decided  to  apply  the  operation  to 
man. 

"  The  symptoms  produced  by  this  operation  have  been 
described  iu  several  scientific  magazines.  The  method 
has  been  tried  at  Paris,  at  Cherbourg-,  and  at  Moscow, 
on  about  fifty  persons  of  both  sexes,  between  the  ages  of 
nineteen  and  sixty-eight,  of  French,  Swiss,  Bussian,  Eng- 
lish, and  American  nationality. 

"  In  every  case  the  method  has  shown  itself  absolutely 
harmless  to  health,  and  the  symptoms  that  it  evoked 
were  a  rise  of  temj^erature,  a  local  sensitiveness  at  the 
place  of  inoculation,  and  the  formation  of  a  transitory 
oedema  at  the  same  iDlace.  The  first  sensations  are  felt 
about  two  or  three  hours  after  inoculation ;  fever  and 
general  indisposition  disappear  after  twenty  -  four  to 
thirty-six  hours  ;  the  sensitiveness  and  oedema  last,  grad- 
ually dying  away  in  from  three  to  four  days.  The  symp- 
toms following  the  second  inoculation  were  generally 
rather  more  marked,  but  of  shorter  diiration.  The  whole 
recalls  the  sensation  of  a  bad  cold  in  the  head,  lasting 
about  one  or  two  days. 

"  The  microbes  introduced  under  the  skin  do  not  jirop- 
agate,  but  after  a  certain  time  they  die  and  disaj)pear. 
It  is  the  substances  which  they  contain,  and  which  are 
set  free  when  they  die,  that  act  upon  the  animal  organ- 
ism and  confer  immunity  upon  it.  It  is  found  that  the 
same  result  can  be  obtained  if  the  microbe  be  killed  be- 
fore inoculation,  and  if  their  dead  bodies  only  be  injected. 
Thus  I  have  been  able  to  prepare  vaccines  preserved  in 
weak  solutions  of  carbolic  acid.  In  this  the  microbes 
die  at  the  end  of  several  hours,  and  the  vaccine  so  pre- 
pared has  been  found  still  efficacious  sis  months  after  its 
preparation.  It  is  evident  that  there  is  much  advantage 
in  this  state  of  preservation  of  the  microbes.  They  can 
be  used  by  persons  having  no  bacteriological  training, 
and  the  absence  of  every  living  organism  makes  them 
perfectly  safe.  The  carbolic  acid  that  they  contain  pre- 
serves them  against  any    invasion  of  other   microbes. 


CHOLERA.  135 

Finally,  as  tliey  can  be  kept  for  several  months,  their 
preparation  can  be  intrusted  to  a  central  laboratory, 
whence  the  vaccine  mn%n)%iles  can  be  sent  out  to  oper- 
ators. But  it  may  be  presumed  that  immunity  given  by 
these  preserved  vaccines  will  not  equal  in  jDersistency 
that  produced  by  living-  ones,  and  as  the  method  is  not 
yet  backed  up  by  established  statistics,  it  is  better  that 
vaccinations  should  be  done  as  much  as  possible  with 
living  virus,  so  as  to  obtain  the  most  conclusive  results. 

"  As  to  the  length  of  time  that  immunity  produced  by 
living  vaccine  lasts,  we  have  not  yet  at  the  laboratory 
animals  that  have  been  inoculated  at  a  very  distant  date ; 
those  upon  which  we  experimented  dated  from,  at  most, 
four  months  and  a  half.  At  the  end  of  this  time  their 
immunity  was  found  to  be  still  perfect,  and  we  do  not 
despair  of  its  lasting  much  longer  yet. 


HARMLESSNESS  OF  THE  METHOD. 

"The  inoculations  upon  man,  added  to  the  hundreds 
of  experiments  that  we  have  made  upon  animals,  testify 
to  the  perfect  harmlessness  of  these  operations,  and  there 
is  no  difficulty  in  proving  their  efficacy  by  experiment, 
no  matter  on  what  species  of  animal.  We  have  taken 
twelve  guinea-pigs,  and  vaccinated  six  of  them  with  vac- 
cines preserved  in  carbolic  acid  since  September  8th  last. 
Yesterday,  at  five  o'clock,  six  days  after  the  first  vaccina- 
tion, we  injected  into  the  peritoneal  cavity  of  all  the  non- 
vaccinated  animals  a  fatal  dose  of  virus,  and  into  the 
vaccinated  animals  we  injected  a  double  dos^  The  six 
vaccinated  animals  are  perfectly  well,  while  of  the  others 
two  have  already  died  of  choleraic  poisoning,  two  are 
very  ill,  and  the  others  will  certainly  soon  become  so. 
But  it  is  evident  that  T  cannot  perform  a  like  experi- 
ment on  man  (but,  however,  this  would  be  the  only  means 
C)i  being  able  to  give  a  definite  experimental  demonstra- 
tion)." 


136  IMMUNITY   AND   SERUM-TIIERAPY. 

Further  details  as  to  the  method  are  given  by  Wood- 
head  in  the  "  Edmburg-h  Hospital  Reports,"  as  follows  : 

"  In  order  to  be  absolutely  certain  that  the  virus  is 
pure,  M.  Hafi'kiue  makes  cultivations  before  each  inocu- 
lation of  the  human  subject,  by  Eoux  and  Yersin's  method, 
one  devised  for  the  separation  of  the  diphtheria  bacillus. 
A  small  drop  of  the  virus  exalte  is  taken  on  a  spatula- 
shaped  needle,  and  streak  after  streak  is  made  with  the 
Hat  of  this  needle  on  the  surface  of  the  agar  in  the  tubes, 
a  couple  of  tubes  being  used,  so  that  twelve  streaks  per- 
haps, in  all,  are  made  without  the  needle  being  recharged  ; 
in  the  earlier  streaks,  of  course,  the  seed  bacilli  are  so 
close  together  that  a  continuous  line  of  colonies  makes 
its  appearance  ;  but  along  the  course  of  the  later  streaks, 
colonies,  with  distinct  intervals  between  them,  are  devel- 
oped ;  part  of  one  of  these  is  examined  under  the  micro- 
scope, in  order  to  determine  that  it  is  made  up  only  of 
comma  bacilli,  and  then  the  other  part  is  used  for  seed 
material  for  a  tube-culture  preparatory  to  inoculation. 

"  The  inoculation  itself  is  an  exceedingly  simple  proc- 
ess ;  the  needle  and  the  syringe  are  boiled ;  the  tube 
containing  the  material  to  be  used  for  inoculation  re- 
ceives a  syringeful  or  pipetteful  of  sterilized  beef-broth, 
then  with  a  platinum  needle  the  culture  is  thoroughly 
mixed  with  this  broth,  so  that  a  kind  of  emulsion  is  pre- 
pared ;  this  emulsion  is  drawn  up  in  a  sterilized  pipette, 
and  is  then  passed  into  a  sterilized  conical  glass  covered 
with  sterilized  paper.  If  a  sixth  of  the  culture  is  to  be 
introduced,  two  more  syringefuls  or  pipettefuls  of  broth 
are  to  be  added,  so  that  we  now  have  three  in  all ;  if  an 
eighth,  three  are  added,  and  so  on  ;  the  whole  is  mixed, 
and  then  half  a  syringeful  is  taken  for  use  for  each  pa- 
tient. In  inoculating,  the  skin,  just  above  the  crest  of  the 
ilium,  is  thoroughly  cleansed  with  five  per  cent,  solution  of 
carbolic  acid,  the  attenuated  virus  is  inoculated  on  the  left 
side,  and  then  after  an  interval  of  four  or  five  daj's  the 
second  vaccine,  or  the  more  virulent  form,  is  inoculated 


CHOLERA.  137 

on  the  right  side.  After  inoculation  everything  that  has 
been  used  is  thoroug-hly  boiled,  the  skin  of  the  patient  is 
again  washed  with  five  per  cent,  carbolic  acid,  and  the  table 
is  washed  down  with  the  same  solution." 

Haffkine  commenced  his  experiments  on  man  by  inocu- 
lating himself,  and  has  repeated  the  inoculation  three 
times.  He  next  inoculated  about  fifty  individuals  in 
Paris,  Cherbourg,  and  Moscow,  and  demonstrated  in  a 
satisfactory  way  that  the  inoculations  are  without  danger. 

A  first  inoculation  in  an  unprotected  person  is  said  to 
give  rise  to  some  malaise  and  febrile  reaction,  to  pain 
and  tumefaction  at  the  point  of  inoculation,  and  swelling 
of  the  neighboring  glands.  The  second  inoculation  with 
a  strong  virus,  made  after  an  interval  of  six  days,  causes 
also  some  elevation  of  temperature,  but  no  swelling  at 
the  point  of  inoculation.  This  slight  reaction  from  a 
strong  virus  is  supposed  to  be  satisfactory  evidence  of  a 
certain  degree  of  immunity  as  a  result  of  the  first  inocu- 
lation. 

The  results  of  the  protective  inoculations  by  Haffkine's 
method,  which  have  been  ijractised  in  India,  indicate 
that  these  inoculations  have  a  real  value,  but  that  immu- 
nity is  not  immediately  established,  and  consequently 
that  during  an  epidemic  a  certain  number  of  fatal  cases 
may  be  expected  among  the  inoculated  as  Avell  as  among 
the  non-inoculated.  This  is  illustrated  by  the  results  of 
inoculations  made  among  the  prisoners  in  Gaya  jail 
(1894),  reported  by  Surgeon-Major  Macrae,  I.M.S.,  from 
whose  report  we  quote  as  follows : 

"  Cholera  broke  out  in  the  Gaya  jail  on  the  9th  of 
July,  and  from  that  date  to  the  2d  August  34  cases  oc- 
curred, with  20  deaths,  there  being  on  date  of  first  attack 
422  prisoners  in  jail.     The  disease  was  clearly  traceable 


t 
138  IMMUNITY   ATSTD   SERUM-THERAPY. 

to  importation,  but  its  diffusion  among  the  prisoners  was 
a  question  of  much  greater  difficulty.  The  sanitary  con- 
dition of  the  jail  is  excellent ;  it  was  built  quite  recently, 
on  the  latest  plans,  and  is  generally  considered  a  model 
jail.  The  water-supply,  which  is  from  a  well,  is  of  excel- 
lent quality  and  protected  from  pollution,  and  it  is  be- 
lieved that  the  spread  of  the  disease  was  largely  due  to 
the  agency  of  flies  finding  access  to  food  and  milk  after 
being  in  contact  with  cholera  poison,  and  contaminating 
them.  From  the  9th  to  the  17th  July,  6  cases  occurred, 
with  5  deaths. 

"  Many  of  the  prisoners  on  being  told  about  ])reventive 
inoculation  wished  to  be  inoculated,  and  M.  Haffkine, 
who  had  previously  been  communicated  with,  and  whose 
zeal  and  enthusiasm  in  the  cause  that  he  so  well  advo- 
cates are  beyond  praise,  arrived  here  on  the  18th  July, 
and  in  the  presence  of  Surgeon-Colonel  Harvey,  who 
kindly  assisted,  and  myself,  inoculated  147  prisoners,  and 
on  the  19th  68,  making  a  total  of  215  out  of  433  present  in 
the  jail  on  that  date. 

"  Being  purely  voluntary,  no  selection  of  prisoners  was 
possible  ;  but  all  classes  in  the  jail  were  represented,  male 
and  female,  old  and  young,  habituals  and  less  frequent 
oftenders,  strong  and  weakly,  convalescent  and  even  hos- 
pital patients  sent  their  representatives.  No  difference 
of  any  kind  was  made  between  inoculated  and  non-inocu- 
lated prisoners  ;  they  were  under  absolutely  identical 
conditions  as  regards  food,  water,  accommodation,  etc., 
in  short,  in  every  possible  respect. 

"  As,  owing  to  the  progress  of  the  epidemic,  a  large 
number  of  prisoners  were  removed  from  the  jail  into 
camp,  it  will  be  found  convenient  to  consider  the  effect 
produced  by  the  anticholera  inoculation  under  three 
headings  : 

"  (a)  The  first  will  include  the  period  from  the  IStli 
July,  the  date  of  first  inoculations,  to  the  24th  July,  the 
date  on  which  final  reinoculations  were  made,  and  refers 
to  all  the  prisoners. 


CHOLERA. 


139 


"  (h)  The  second  concerns  the  prisoners  who  remained 
in  jail  after  the  majority  were  removed  into  camp,  and 
comprises  the  period  from  the  25th  July,  to  the  2d  August, 
on  which  date  the  final  case  occurred  among'  this  body  of 
prisoners. 

"(c)  The  tldrd  refers  to  the  body  of  prisoners  who 
were  moved  into  camp  on  the  25th  July,  and  includes  the 
period  between  that  date  and  the  1st  Aug"ust,  when  the 
final  case  occurred  among  this  body. 


Averase 
present. 

Chol- 
era. 

Percent- 
age of 
average 

strength. 

Deaths. 

Percent- 
age of 
average 

strength. 

Percent- 
age of 
deaths  to 
cases. 

Inoculated 

211.2 
209.0 

32.5 
48.55 

171.42 
146.5 

5 

7 

1 

7 

2 
6 

No.  I. 
2.37 
3.34 

No.  II. 
3.07 

14.42 

No.  III. 
1.16 
4.09 

4 
5 

Nil. 
3 

1 
2 

1.89 
2.39 

Nil. 
6.18 

0.58 
1.36 

80  0 

Not  inoculated 

Inoculated 

71.42 
Nil. 

Not  inoculated 

Inoculated 

42.86 
50.0 

Not  inoculated 

33.33 

"  The  conclusions  to  be  drawn  from  the  results  above 
recorded  appear  to  me  to  be  that  for  the  first  few  days 
the  inoculations  have  scarcely  any  protective  influence ; 
then  their  effect  seems  to  gradually  increase.  M.  Haff- 
kine  in  his  publications  has  laid  stress  on  the  fact  that 
he  anticipates  a  period  of  ten  days  would  elapse  from 
date  of  first  inoculations  before  the  full  effect  would  be 
obtained. 


During  the  first 
five  days  after 
fi  r  s  t    inocula- 
TION. 

First     three 
dats      after 
second     inocu- 
LATION. 

Last   six  days. 

Cases. 

Deaths. 

Cases. 

Deaths. 

Cases. 

Deaths. 

Inoculated 

5 

7 

4 
5 

3 
5 

1 
3 

Nil. 
8 

Nil. 

Not  inoculated 

2 

140 


IMMUNITY    AND   SERTTM-TIIEKAPY. 


"  Further  observations  are  necessary  to  prove  whether 
the  inoculations  as  now  practised  will  prove  of  lasting 
benelit ;  the  results  obtained  in  Gaya  jail  seem  to  me  to 
justify  the  conclusion  that  their  temporary  beneficial  ef- 
fect is  undoubted. 

"I  have  been  informed  by  M.  Haffkine  that  he  pro- 
poses to  introduce  a  certain  modification  of  his  method, 
with  the  object  of  afibrding-  protection  to  patients  during 
the  ten  days  necessary  for  the  action  of  his  vaccines.  I 
think  there  is  every  reason  to  believe  that  better  results 
would  have  been  obtained  here  had  the  inoculations 
been  performed  at  an  earlier  period  instead  of  during  the 
epidemic." 

In  a  recent  pajaer  {British  Medical  Journal,  January 
26,  1895)  Haft'kine  gives  the  following  summary  of  his 
inoculations  in  India : 

"  Table  showing  the  total  7iumher  of  persons  on  whom  ob- 
servations have  been  made  in  Calcutta,  Gaya,  Cawnj^ore, 
ccnd  L'uchtiow. 


Number. 

Cases. 

Percentage 

of  Cases 

to  Strength. 

Percentage 

Deaths.         of  Deaths 

to  Strength. 

Non-inoculated. 
Inoculated 

1,735 
500 

174 
21 

10.63 
4.20 

113     !        6.51 
19             3.80 

Total 

2,235 

195 

132 

Other  methods  of  producing  immunity  in  man  have 
been  proposed,  and  recent  experiments  indicate  that  this 
may  be  accomplished  through  the  digestive  tract  by  the 
ingestion  of  considerable  quantities  of  sterilized  cultures. 
Thus  Klemperer  (1892)  has  obtained  results  which  seem 
to  show  that  immunity  in  man  may  be  induced  not  only 
by  the  subcutaneous  injection  of  virulent  cultures  but 
also  by  the  subcutaneous  injection  of  the  milk  of  immu- 


CHOLERA.  141 

nized  goats  and  by  the  ingestion  of  cultures  sterilized  by 
heat.  The  degree  of  immunity,  as  determined  by  the 
activity  of  the  blood-serum  of  the  immune  individual  for 
the  protection  of  guinea-pigs,  is  considerably  less,  how- 
ever, than  when  repeated  injections  of  virulent  cultures 
have  been  made.  The  blood-serum  of  individuals  made 
immune  by  the  last-mentioned  method  is  said  by  Klemp- 
erer  to  protect  guinea-pigs  when  injected  into  the  cavity 
of  the  abdomen  in  the  dose  of  0.005  c.c.  And  the  injec- 
tion of  5  c.c.  of  milk  from  an  immunized  goat  is  said  to 
confer  such  an  immunity  that  0.25  c.c.  of  blood-serum 
from  the  immune  individual  is  sufficient  to  protect  a 
guinea-pig  from  cholera  cultures.  In  this  connection 
attention  is  called  to  the  results  obtained  in  more  recent 
experiments  by  Pfeiffer  (p.  118). 

Sawtschenko  and  Sabolotny  (1893),  as  a  result  of  a  se- 
ries of  experiments  made  upon  themselves  and  laboratory 
assistants,  arrive  at  the  following  conclusions  : 

"  1.  After  the  ingestion  of  sterilized  (by  heat)  and  sub- 
sequently carbolized  agar  cultures  of  cholera  bacteria 
the  serum  of  man  acquires  an  immunizing  property  as 
regards  the  cholera  vibrio. 

"  2.  As  a  result  of  the  ingestion  of  sterilized  agar  cult- 
ures the  individual  is  protected  from  infection  with  viru- 
lent cultures  of  the  cholera  vibrio  by  way  of  the  intestine. 

"  3.  The  discharges  of  individuals  immune  against  chol- 
era, and  to  all  outward  appearance  in  perfect  health,  may 
contain  a  great  number  of  cholera  vibrios  (in  case  they 
are  in  any  way  introduced  into  the  intestine)  and  may 
thus  serve  to  propagate  the  malady." 

SeruT^i-thcrapy . 

The  results  of  experiments  already  referred  to  are  fa- 
vorable to  the  view  that  the  serum  of  immune  individuals 


142  IMMUNITY    AND   SERUM-THEllAPT. 

(man  or  lower  animals)  contains  a  substance  (antitoxin  ?) 
wliicli  would  i^robably  be  useful  in  the  treatment  of  chol- 
era if  a  practical  method  of  obtaining-  it  in  sufficient 
quantit}^  should  be  devised.  This  view  receives  support 
from  the  exj)eriments  of  Pawlowsky  and  Buchstab  (1893) 
and  of  Fedoroff  (1893).  The  first-named  bacteriologists, 
after  demonstrating  the  fact  that  guinea-pigs,  rabbits,  and 
dogs  could  readily  be  immunized  by  the  methods  hereto- 
fore refen*ed  to,  proceeded  to  make  further  experiments 
with  the  serum  of  immunized  dogs.  The  serum  from 
these  animals  proved  to  have  a  decided  bactericidal  ac- 
tion for  cholera  cultures,  and  when  injected  into  the  peri- 
toneal cavity  of  rabbits  or  guinea-pigs,  in  doses  of  5  c.c, 
it  made  these  animals  immune  against  the  usually  fatal 
doses  of  virulent  cultures  of  the  cholera  spirillum.  The 
dogs  were  immunized  by  first  injecting  attenuated  cult- 
ures subcutaneously  in  doses  of  10  c.c,  and  in  following' 
this  with  more  virulent  cultures  and  larger  doses,  until 
at  last  the  animal  received  90  c.c.  of  a  virulent  culture. 
The  immunizing  value  of  the  serum  obtained  from  a  dog 
treated  in  this  way  was  estimated  at  1  to  130,000.  Five  c.c. 
of  a  virulent  culture  mixed  with  1  c.c.  of  this  serum  pro- 
duced no  efi'ect  when  injected  into  the  peritoneal  cavity 
of  guinea-pigs.  And  the  curative  value  of  the  serum, 
w'hen  injected  subcutaneously  two  to  five  hours  after  in- 
traperitoneal infection  with  5  c.c.  of  a  virulent  culture, 
was  shown  b}^  the  fact  that  out  of  16  animals  treated  in 
this  way  12  survived,  while  the  control  animals  all  died. 
The  serum  was  injected  by  the  authors  referred  to  be- 
neath their  own  skin  in  doses  of  1  c.c,  and  proved  to  have 
no  noticeable  effect  upon  the  pulse  or  temperature. 

Fedorofl*  (1893)  did  not  succeed  in  obtaining  a  serum  of 
as  high  an  immunizing-  value  as  that  obtained  by  the  au- 


CHOLERA.  143 

thors  last  referred  to,  or  by  Klemperer,  but  he  arrives  at 
the  conclusion  that  "  it  is  a  settled  fact  that  animals  may- 
be rendered  immune  against  cholera  in  a  surprisingly 
short  time  by  means  of  the  blood-serum  of  other  immune 
animals.  Also  that  blood-serum  therapy  in  Asiatic  chol- 
era seems  much  more  promising  than  the  method  pro- 
posed by  Brieger,  Kitasato,  and  Wassermann — injections 
of  cultures  in  thymus  bouillon." 

Dr.  Freymuth,  of  Danzig,  in  the  Deutsche  Medicinische 
Woche/ischrift  of  October  25,  1894,  reports  three  cases  of 
cholera  treated  by  subcutaneous  injections  of  blood-serum 
from  individuals  who  had  recently  suffered  attacks. 

The  first  case,  a  female,  was  in  a  complete  state  of  col- 
lapse on  August  13tli,  when  10  c.c.  of  serum  was  injected 
under  the  skin  between  the  shoulders.  No  improvement 
was  noted  as  a  result  of  the  injection.  The  following 
day  a  dose  of  30  c.c.  was  given  in  the  same  way.  No  im- 
provement. The  following  day  a  dose  of  50  c.c.  was  fol- 
lowed by  apparent  improvement,  but  this  was  not  main- 
tained, and  the  patient  died  on  the  18th. 

In  the  other  two  cases  recovery  occurred.  One  received 
in  all  80  c.c.  of  serum,  and  the  other  20.  As  the  reporter 
observes,  the  number  of  cases  is  too  small  to  justify 
any  conclusions  as  to  the  value  of  the  treatment ;  but 
they  are  of  interest  as  being  the  first  cases  reported  in 
which  this  mode  of  treatment  was  resorted  to,  and  as 
showing,  at  least,  that  it  is  harmless  and  not  difficult  to 
carry  out  when  there  are  persons  at  hand  who  have  re- 
covered from  a  recent  attack,  and  are  Avilling  to  spare 
the  blood. 

With  reference  to  the  practical  value  of  protective  in- 
oculations for  the  prevention  of  cholera  epidemics,  the 
writer  sees  no  reason  to  modify  the  views  expressed  in 


144  IMMUNITY   AND   SERUM-TIIEKAPY. 

his  paper  on  "  Protective  Inoculations  in  Infectious  Dis- 
eases," read  at  the  twentieth  annual  meeting  of  the 
American  Public  Health  Association  (1892).  Speaking 
of  Ferrdn's  inoculations,  and  the  more  recent  exioeriments 
of  Haffkine,  I  say : 

"Whether  this  method  will  be  found  to  have  any  great 
practical  value,  can  only  be  determined  by  more  extended 
exiDeriments.  But  in  view  of  the  fact  that  other  measures 
of  prophylaxis,  well  known  to  sanitarians,  are  sufficient 
for  the  prevention  of  cholera  epidemics,  and  that  nurses 
and  others  who  necessarily  come  in  contact  with  cholera 
patients  are  not  likely  to  contract  the  disease  if  they  use 
proper  precautions  with  reference  to  their  food  and  drink, 
the  disinfection  of  their  hands,  etc.,  we  doubt  whethei 
protective  inoculations  will  ever  come  into  general  use 
as  a  measure  of  prophylaxis  against  this  disease.  Cer- 
tainly they  cannot  take  the  place  of  those  sanitary  meas- 
ures which  have  been  proved  to  be  sufficient  for  the  pre- 
vention of  epidemics,  namely,  exclusion  by  a  proper 
inspection  service  at  ports  of  entry  ('  quarantine '),  isola- 
tion of  the  sick,  disinfection  of  excreta,  general  sanitary 
police  of  exposed  towns  and  cities,  boiling  the  water  used 
for  drinking  purposes,  etc.  Still,  under  certain  circum- 
stances, i^rotective  inoculations  may  have  considerable 
practical  importance,  and  the  experiments  now  being 
made  have  evidently  great  scientific  interest  in  connection 
with  the  question  of  acquired  immunity." 


BIBLIOGRAPHY. 

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CHOLERA.  146 

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10 


146  IMMUNITY   AND   SERUM-THERAPY. 

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schweinchen  gegeu  den  Vibrio  Ivanoff.  Zcitsclir.  fiir  Hyg.,  XVII., 
117,  1894. 

Jaweia,  G.  :  Observations  sur  des  cobayes  immunises  par  les  vaccins 
anticholeriques  vivants.     Annal.  de  I'lust.  Pasteur,  1892,  p.  708. 

Kanthack  and  Westbrook  :  Report  on  Immunity  against  Cholera.  Brit- 
ish Medical  Journal,  1893,  p.  572. 

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Klein,  E.  :  The  Anticholera  Vaccination  ;  an  Experimental  Critique. 
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Medical  Gazette,  1893,  p.  135.  ;  Ibid.,  p.  138.  (Klein's  critique  criti- 
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Untersuchunaren   iiber  Infektiou   und  Immunitat  bei  der  asia- 


tischen  Cholera,  8°,  p.  183.     Berlin  (August  Hirschwald),  1894. 

Lazarus,  A.  :  Ueber  antitoxisclie  Wirksamkeit  des  Blutserums  Cliolera- 
geheilter.     Berl.  klin.  Wchschr.,  1892,  p.  1041. 

Macrae,  R.  :  Preventive  Inoculation  for  Cholera  in  India.  British  Med- 
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Cholera  and  Preventive  Inoculation  in  Gaya  Jail.  Indian  Medi- 
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CHOLERA.  147 

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IV. 

DIPHTHERIA. 

Diphtheria  is  g-enerally  recognized  by  physicians  as  a 
specific  infectious  disease,  and,  owing  to  its  wide  preva- 
lence and  fatal  character,  a  precise  knowledge  of  its  eti- 
ology is  of  the  greatest  importance.  Until,  as  a  result  of 
recent  researches,  this  was  determined,  pathologists  were 
in  doubt  as  to  whether  diphtheria  should  be  considered 
as  primarily  a  local  infection,  or  whether  the  local  mani- 
festations were  secondary  to  a  general  systemic  infec- 
tion. But  this  question  appears  now  to  be  definitely 
settled  in  favor  of  the  former  view.  We  have  to-day  a 
very  precise  knowledge  of  the  specific  infecting  agent, 
and  have  evidence  that  it  produces  during  its  growth  a 
very  potent  toxic  substance,  the  absorption  of  which 
from  the  seat  of  local  infection  accounts  in  a  satisfactory 
manner  for  the  general  symptoms  of  the  disease,  which 
are  due  to  toxaemia  and  not  to  an  invasion  of  the  blood 
and  tissues  by  the  pathogenic  microorganism  produc- 
ing it. 

Numerous  researches  by  competent  bacteriologists 
have  failed  to  demonstrate  the  presence  of  bacteria  in 
the  blood  of  patients  suffering  from  diphtheria,  but 
various  microorganisms  have  been  obtained  in  cultures 
from  diphtheritic  pseudo-membranes,  and  may  be  demon- 
strated by  the  microscopical  examination  of  stained  prep- 
arations.    Among  these  are  the  well-known  pus  organ- 


DIPHTHEEIA.  149 

isms,  and  especially  tlie  streptococcus  pyogenes,  wliicli 
appears  to  be  very  commonly  present,  and  is  the  active 
agent  in  the  production  of  certain  forms  of  pseudo-diph- 
theria. But  the  malig-nant,  specific  diphtheria,  so  well 
known  in  this  country  and  in  Europe,  has  been  demon- 
strated by  the  recent  researches  of  bacteriologists  to  be 
due  to  a  bacillus,  first  recognized  by  Klebs  in  stained 
preparations  of  diphtheritic  false  membranes  (1883),  and 
cultivated  and  described  by  Lofiier  in  1884.  In  his  first 
publication  Loffler  did  not  claim  to  have  fully  demon- 
strated the  etiological  relation  of  this  bacillus,  but  this 
appears  to  be  fully  established  by  subsequent  researches. 

In  his  first  research  Lofiier  studied  twenty -five  cases, 
and  in  the  greater  number  of  them  found  in  stained 
preparations  the  bacillus  previously  described  by  Klebs. 
From  six  of  these  cases  he  obtained  it  in  pure  cultures, 
and  by  experiments  on  pigeons,  chickens,  rabbits,  and 
guinea-pigs  proved  that  it  gave  rise  to  a  diphtheritic  in- 
flammation when  inoculated  into  the  mucous  membrane  of 
the  trachea,  conjunctiva,  pharynx,  or  vagina.  In  a  second 
communication  Lofiier  reported  his  success  in  finding  the 
same  bacillus  in  ten  additional  cases,  and  also  that  he 
had  isolated  from  the  same  source  a  nou -pathogenic  ba- 
cillus which  resembled  it  very  closely.  This  pseudo- 
diphtheria  bacillus  has  since  been  found  by  other  bacte- 
riologists (Von  Hoffman,  Roux  and  Yersin),  and  it  is 
uncertain  whether  it  is  to  be  considered  a  distinct  spe- 
cies, or  a  non-pathogenic  variety  of  the  diphtheria  bacil- 
lus as  maintained  by  Eoux  and  Yersin.  But  its  occasional 
presence  does  not  invalidate  the  very  positive  experi- 
mental evidence  relating  to  the  specific  pathogenic  power 
of  the  true  diphtheria  bacillus. 

According  to  Roux  and  Yersin,  "  attenuated  varieties  " 


150  IMMUNITY   AND   SERUM-THERAPY. 

of  the  diphtheria  bacillus  may  be  obtained  by  cultivating- 
it  at  a  temperature  of  39.5"  to  40°  C.  in  a  current  of  air ; 
and  these  authors  suggest  that  a  similar  attenuation  of 
pathogenic  power  may  occur  in  the  fauces  of  convales- 
cents from  the  disease,  and  that  possibly  the  similar 
non-pathogenic  bacilli  which  have  been  described  by 
various  investigators  have  originated  in  this  v^ay  from  the 
true  diphtheria  bacillus.  These  authors  further  state, 
in  favor  of  this  view,  that  from  diphtheritic  false  mem- 
brane, preserved  by  them  in  a  desiccated  condition  for 
five  months,  they  obtained  numerous  colonies  of  the  ba- 
cillus in  question,  but  that  the  cultures  were  destitute  of 
pathogenic  virulence.     They  say : 

"  It  is  then  possible,  by  commencing  with  a  virulent 
bacillus  of  diphtheria,  to  obtain  artificially  a  bacillus  with- 
out virulence,  quite  similar  to  the  attenuated  bacilli 
wdiich  may  be  obtained  from  a  benign  diphtheritic  an- 
gina, or  even  from  the  mouth  of  certain  persons  in  good 
health.  This  microbe,  obtained  aiiificiallj^,  resembles 
completely  the  pseudo-diphtheritic  bacillus  ;  like  it,  it 
grows  more  abundantly  at  a  low  temperature  ;  it  renders 
bouillon  more  rapidly  alkaline  ;  it  grows  with  difficulty 
in  the  absence  of  oxygen." 

Subcutaneous  inoculations  in  guinea-pigs  of  a  small 
quantity  of  a  pure  culture  of  the  bacillus  (0.1  to  0.5  c.c.  of 
a  bouillon  culture)  cause  death  in  from  one  to  four  or 
five  days.  The  usual  changes  observed  at  the  autopsy 
are — 

"  An  extensive  local  oedema,  with  more  or  less  hyper- 
emia and  ecchymosis  at  the  site  of  inoculation,  fre- 
quently swollen  and  reddened  lymphatic  glands,  in- 
creased serous  fluid  in  the  peritoneum,  pleura,  and 
pericardium,  enlarged  and  hemorrhagic  suprarenal  cap- 


DIPHTHERIA.  151 

sules,  occasionally  slightly  swollen  spleen,  sometimes 
fatty  degenerations  in  the  liver,  kidney,  and  myocardium. 
We  have  always  found  the  Liiffler  bacilli  at  the  seat  of 
inoculation  most  abundant  in  a  grayish-white,  librino- 
purulent  exudate  present  at  the  point  of  inoculation,  and 
becoming  fewer  at  a  distance  from  this,  so  that  the  more 
remote  'parts  of  the  cedematous  fluid  do  not  contain  any 
bacilli  "  (Welch  and  Abbott). 

The  authors  quoted  agree  with  Liiffler  and  others  in 
stating  that  the  bacillus  is  only  found  at  the  point  of  in- 
oculation. In  all  cases  their  cultures  from  the  blood  and 
from  the  various  organs  gave  a  negative  result. 

Rabbits  are  not  so  susceptible,  and  may  recover  after 
the  subcutaneous  inoculation  of  very  small  doses,  but 
usually  die  in  from  four  to  twenty  days  when  2  to  4  c.c. 
of  a  bouillon  culture  have  been  introduced  beneath  the 
skin.  In  these  animals,  also,  there  is  an  extensive  local 
cedema,  enlargement  of  the  neighboring  lymphatic 
glands,  and  a  fatty  degeneration  of  the  liver.  Eoux  and 
Yersin  have  shown  that  in  rabbits,  w^hen  death  does  not 
ensue  too  quickly,  paralysis  of  the  posterior  extremities 
frequently  occurs,  thus  completing  the  experimental 
proof  of  the  specific  pathogenic  power  of  pure  cultures 
of  this  bacillus. 

Similar  symptoms  are  produced  in  pigeons  by  the  sub- 
cutaneous inoculation  of  0.5  c.c.  or  more,  but  they  com- 
monly recover  when  the  quantity  is  reduced  to  0.2  c.c. 
(Roux  and  Yersin), 

The  rat  and  the  mouse  have  a  remarkable  immunity 
from  the  eflfects  of  this  poison.  Thus,  according  to  Roux 
and  Yersin,  a  dose  of  2  c.c,  which  would  kill  in  sixty 
hours  a  rabbit  weighing  3  kilogr.,  is  without  effect  upon 
a  mouse  which  weighs  only  10  gm. 


152  IMMUNITY    AND   SERUM-TIIERAPY. 

Old  cultures  are  somewhat  less  yirulent  than  fresh 
ones,  but  when  replanted  in  a  fresh  culture  medium  they 
manifest  their  original  yirulcnce.  Thus  a  culture  upon 
blood-serum  which  was  five  months  old  was  found  by 
Roux  and  Yersin  to  kill  a  guinea-pig-  in  five  days,  but 
when  replanted  it  killed  a  second  animal  of  the  same 
species  in  twenty-four  hours. 

Evidently  a  microorganism  which  destroys  the  life  of  a 
susceptible  animal  when  injected  beneath  its  skin  in 
small  quantity,  and  which  nevertheless  is  only  found  in 
the  vicinity  of  the  point  of  inoculation,  must  owe  its 
pathogenic  power  to  the  formation  of  some  potent  toxic 
substance,  which,  being  absorbed,  gives  rise  to  toxaemia 
and  death.  This  inference  in  the  case  of  the  diphtheria 
bacillus  is  fully  sustained  by  the  results  of  recent  exper- 
imental investigations.  Eoux  and  Yersin  (1888)  first 
demonstrated  the  pathogenic  power  of  cultures  which 
had  been  filtered  through  porous  porcelain.  Old  cultures 
were  found  by  these  experimenters  to  contain  more  of 
the  toxic  substance  than  recent  ones,  and  to  cause  the 
death  of  a  guinea-pig  in  a  dose  of  2  c.c.  in  less  than 
twenty-four  hours.  The  filtered  cultures  produced  in 
these  animals  the  same  eflfects  as  those  containing  the 
bacilli — local  cedema,  hemorrhagic  congestion  of  the  or- 
gans, effusion  into  the  pleural  cavity.  Somewhat  larger 
doses  were  fatal  to  rabbits,  and  a  few  drops  injected  sub- 
cutaneously  sufficed  to  kill  a  small  bird  within  a  few 
hours.  In  their  second  paper  (1889)  the  authors  men- 
tioned state  that  so  long  as  the  reaction  of  a  culture  in 
bouillon  is  acid,  its  toxic  power  is  comparatively  slight, 
but  that  in  old  cultures  the  reaction  is  alkaline,  and  in 
these  the  toxic  potency  is  greatly  augmented.  With  such 
a  culture,  filtered  after  having  been  kept  for  thirty  days, 


DIPHTHEKIA.  153 

a  dose  of  one-eightli  of  a  cubic  centimetre  injected  sub- 
cutaneously,  sufficed  to  kill  a  g-uinea-pig- ;  and  in  larger 
amounts  it  proved  to  be  fatal  to  dog-s  when  injected 
directly  into  the  circulation  through  a  vein. 

The  same  authors,  in  discussing  the  nature  of  the  poi- 
son in  their  filtered  cultures,  infer  that  it  is  related  to 
the  diastases,  and  state  that  its  toxic  potency  is  very 
much  reduced  by  exposure  to  a  comparatively  low  tem- 
perature— 58°  C,  for  two  hours — and  completely  de- 
stroyed by  the  boiling-  temperature — 100°  C,  for  twenty 
minutes.  It  was  found  to  be  insoluble  in  alcohol,  and 
the  precipitate  obtained  by  adding-  alcohol  to  an  old 
culture  proved  to  contain  the  toxic  substance.  Loffler 
also  has  obtained,  by  adding-  five  volumes  of  alcohol  to 
one  of  a  pure  culture,  a  white  precipitate,  soluble  in 
water,  which  killed  rabbits  in  the  dose  of  0.1  to  0.2  gva. 
when  injected  beneath  the  skin  of  these  animals.  It 
g-ave  rise  to  a  local  oedema  and  necrosis  of  the  skin  in  the 
vicinity  of  the  point  of  inoculation,  and  to  hyper?emia  of 
the  internal  organs.  This  deadly  foxhi  appears  to  be  an 
albuminoid  substance,  but  its  exact  chemical  composition 
has  not  yet  been  determined. 

Brieg-er  and  Frankel  (1891)  obtained  results  correspond- 
ing with  those  previously  reported  by  Eoux  and  Yersin. 
Their  researches  showed  that  the  toxic  substance  con- 
tained in  diphtheria  cultures  is  destroyed  by  a  tempera- 
ture of  60°  C.  ;  that  it  is  soluble  in  water,  and  insoluble 
in  alcohol ;  that  it  does  not  pass  through  a  dialyzing 
membrane,  and  has  not  the  chemical  characters  of  the 
ptomaines  or  toxins,  but  is  an  albuminous  body — a  toxal- 
bumin.  It  was  obtained  by  the  authors  named  b}^  pre- 
cipitation with  slightly  acidified  (acetic  acid)  alcohol ; 
the  precipitate,  after  being  washed  in  a  dialyzer  and  dried 


154  I^IMUNITY   AND   SERUM-THERAPY. 

• 

in  a  vacuum  at  a  temperature'of  40"  C,  was  a  snow-white, 
amorphous,  crnmblin"-  mass. 

Wasscrmann  and  Proskauer  (1892)  found  that  the  alco- 
holic precipitate  from  diphtheria  cultures  contains  two 
diiferent  substances,  which  are  disting-uished  by  their  dif- 
ferent degrees  of  solubility  in  diluted  and  absolute  alco- 
hol ;  both,  however,  give  the  usual  reactions  of  albumi- 
nous bodies,  and  pass  very  slowly  through  a  dialyzing 
membrane.  Only  one  of  these  substances  laossesses  toxic 
properties.  After  the  removal  of  peptone  and  globulin 
from  the  filtered  cultures,  these  were  evaporated  and  a 
precipitate  obtained  of  one  of  the  albuminous  substances 
by  means  of  sixty  per  cent,  to  seventy  per  cent,  alcohol. 
The  other  substance  remained  in  solution,  and  was  sub- 
sequently obtained  by  precipitation  with  absolute  alco- 
hol. The  substance  first  obtained  by  this  method  is 
toxic,  and  the  other  precipitate  is  not.  The  authors 
named  succeeded  in  killing  rabbits  with  the  toxalbumin 
obtained  in  this  way,  but  were  not  able  to  produce  im- 
munity in  these  animals  by  the  injection  of  non-fatal 
doses.  Frankel  (1891)  had  previously  reported  his  faihn-e 
to  immunize  guinea-pigs  by  the  injection  of  the  dry  pre- 
cipitate, obtained  in  his  experiments  from  dii)htheria 
cultures  ;  but  when  filtered  cultures,  or  cultures  sterilized 
by  heat  (55"  C.  for  one  hour),  were  injected  into  these  ani- 
mals, they  showed  an  increased  resistance  to  the  patho- 
genic action  of  virulent  cultures.  Still  better  results 
were  obtained  when  10  c.c.  of  a  bouillon  culture,  heated 
to  100°  C,  were  injected  subcutaneously,  but  still  this 
method  was  not  entirely  reliable.  But  true  immunity 
was  established  by  injecting  into  the  peritoneal  cavity  10 
to  20  c.c.  of  a  bouillon  culture  heated  to  65"  to  70°  C.  for 
one  hour.     The  immunity  was  not  fully  established  until 


DIPHTHEEIA.  155 

about  fourteen  days  after  the  protective  inoculation. 
Friinkel  arrives  at  the  conclusion  that  the  cultures  must 
contain  an  immunizing-  substance  as  well  as  a  toxic  pro- 
teid,  as  the  diphtheria  toxalbumin  is  destroyed  by  the 
temperature  (65°  to  70°  C.)  used  in  the  preparation  of  his 
cultures  for  producing-  immunity. 

Behring-,  in  the  same  year  (1891),  commenced  his  exper- 
iments upon  diphtheria  immunity.  Guinea-pigs  were 
made  immune  by  the  use  of  sterilized  cultures,  and  by 
inoculations  with  virulent  cultures,  four  weeks  old,  to 
which  iodine  terchloride  had  been  added  in  the  propor- 
tion of  1  to  500^ the  mixture  was  allowed  to  stand  for 
sixteen  hours.  Animals  were  also  immunized  by  inject- 
ing beneath  the  skin  a  virulent  culture  of  the  bacillus, 
and  then  treating  them  with  subcutaneous  injections  of 
iodine  terchloride  (2  c.c),  which  was  thrown  under  the 
skin  for  three  days  in  succession  in  the  vicinity  of  the 
point  of  inoculation.  The  guinea-pigs  treated  in  this 
way  remained  sick  for  some  time,  but  finally  recovered 
and  were  subsequently  immune.  Still  better  results 
were  obtained  when  rabbits  were  subjected  to  the  same 
treatment.  The  animals  were  immune  against  the  toxic 
action  of  sterilized  cultures,  as  well  as  against  infection 
by  virulent  diphtheria  bacilli. 

In  subsequent  experiments  (1892)  Behring  and  Wer- 
nicke used  cultures  which  had  been  attenuated  by  con- 
tact with  iodine  terchloride  for  from  thirty-six  to  forty- 
eight  hours,  and  proved  that  the  method  could  be  suc- 
cessfully employed  in  immunizing  sheep ;  and  the  fact 
was  ascertained  that  blood-serum  from  an  immune  ani- 
mal could  be  used  with  success  in  arresting  diphtheritic 
infection  in  susceptible  animals.  To  preserve  the  serum, 
which  they  obtained  from  immunized  sheep,  rabbits,  and 


156  IMMUNITY   AND   SERUM-THERAPY. 

giiinea-pigs,  they  added  to  it  0.5  per  cent,  of  pure  carbolic 
acid.  For  producing-  immunity  tliey  found  that  a  smaller 
amount  of  serum  was  required  than  was  necessary  for  the 
cure  of  an  animal  already  infected.  If  the  injection  was 
made  immediately  after  infection  from  one  and  a  half  to 
two  times  the  amount  was  required ;  eight  hours  after 
infection  the  amount  was  three  times  as  great,  and  twenty- 
four  to  thirty-six  hours  after  infection  the  dose  required 
was  eight  times  the  immunizing  dose. 

The  immunizing  value  of  blood-serum  from  different 
animals  was  estimated  by  finding  the  smallest  dose  which 
would  protect  an  animal  from  fatal  infection  by  the  mini- 
mum lethal  dose  of  a  culture,  the  toxic  potency  of  which 
had  been  carefully  determined.  The  value  is  expressed  in 
figures  which  give  the  proportion  required  comi:)ared  with 
the  body-weight  of  the  animal.  Thus  an  immunizing 
value  of  100  would  mean  that  1  gm.  of  the  serum  is  suffi- 
cient to  protect  an  animal  weighing  100  gm.  from  the  fatal 
effect  which  would  be  produced  in  a  control  animal  of  the 
same  weight  by  infection  with  a  virulent  culture  of  the 
diphtheria  bacillus  in  the  minimum  dose  required  to  pro- 
duce this  result.  The  cultures  employed  are  made  in 
bouillon  containing  one  per  cent,  of  peptone  ;  they  are 
inoculated  from  agar  cultures  and  are  kept  in  the  incu- 
bating oven  for  two  days.  Cultures  prepared  in  this  way 
were  found  to  be  quite  uniform  in  their  pathogenic  viru- 
lence as  tested  upon  guinea-pigs.  But  when  cultures 
are  kept  for  some  time  there  is  an  increase  in  virulence. 
Thus  a  culture  obtained  from  a  fatal  case  of  diphtlieria 
which  in  1890  killed  guinea-pigs  in  three  to  four  days, 
when  injected  subcutaneously  in  the  dose  of  0.1  c.c.  (two 
days  old  bouillon  culture),  at  the  end  of  a  year  was  fatal 
to  these  animals  in  the  dose  of  0.025  c.c.     This  increase 


DIPHTHERIA.  157 

in  virulence  is  ascribed  to  the  fact  that  the  cultures  were 
renewed  at  long-  intervals. 

More  recently  (1894)  Behring  has  lixed  a  standard  for 
what  he  calls  a  normal  therapeutic  serum.  This  is  a 
serum  which  when  injected  into  guinea-pig-s  in  the  pro- 
portion of  1  to  5,000  of  body-weight  saves  the  animal 
from  the  fatal  effects  of  ten  times  the  minimum  dose  of 
a  culture  in  bouillon,  two  days  old,  which  would  kill  a 
control  animal  not  treated. 

In  a  recent  communication  (November,  1894)  Behring 
states  his  conclusion  that  for  producing  immunity  in  man, 
150  normal  antitoxin  units  should  be  given,  instead  of  60 
as  he  had  previously  recommended. 

The  serum  manufactured  under  his  direction  is  said 
(September,  1894)  to  be  of  two  kinds — one,  obtained  from 
the  horse,  has  a  value  of  60  normal  antitoxin  units  ;  the 
other  has  a  value  of  140  units.  Of  the  weaker  serum 
Behring  says  experience  has  demonstrated  that  for  chil- 
dren under  ten  years  of  age  10  c.c.  is  sufficient  to  arrest 
the  progress  of  the  disease  and  effect  a  cure  if  given 
within  two  or  three  days  from  the  outset  of  the  attack. 
For  producing  immunity  in  children  subject  to  infection, 
one-tenth  of  this  amount  (1  c.c.)  is  said  to  be  sufficient. 
Of  the  stronger  serum  1  c.c.  is  sufficient  to  arrest  the 
disease  during  the  incubation  period ;  and,  according  to 
Behring,  out  of  100  cases  treated  during-  the  first  forty- 
eight  hours  with  the  single  therapeutic  dose  (10  c.c.  -of 
serum  having  a  value  of  60  =  600  normal  units)  not  5  will 
die.  The  later  the  treatment  is  commenced  the  larger 
will  be  the  dose  required.  Behring  further  states  that 
the  diphtheria  antitoxin  has  no  injurious  effect  upon 
animals  in  the  largest  doses  that  have  been  employed, 
and  that  aside  from   its   antitoxic  power  its   iiroperties 


158  IMMUNITY   AND   SERUM-THERAPY. 

are  entirely  negative  so  far  as  living  animals  are  con- 
cerned. 

Aronson  (1893),  in  experiments  on  dogs,  succeeded  in 
producing  immunity  by  the  use  of  attenuated  cultures,  or 
of  cultures  to  which  formaldehyd  had  been  added  ;  also 
by  feeding  the  animal  large  quantities  of  diphtheria 
bouillon;  and,  finally,  by  injection  of  the  blood  of  natur- 
ally immune  animals  (white  rats)  into  which  large  quan- 
tities (10  c.c.)  of  a  virulent  culture  had  been  injected. 
Two  months  after  receiving  several  such  injections  it  was 
found  that  0.2  gm.  of  blood-serum  from  the  rat  sufficed 
to  save  a  guinea-i3ig  from  ftital  infection.  In  experiments 
on  dogs  an  immunity  was  established  in  six  weeks  by 
the  injection  of  a  large  amount  of  a  virulent  culture, 
which  had  a  value  of  1  to  30,000,  i.e.,  0,01  c.c.  of  this 
serum  sufficed  to  protect  a  guinea-pig  weighing  300  gm. 
From  100  gm.  of  this  serum  Aronson  claims  to  have  ob- 
tained 0.8  gm.  of  a  substance  which  had  a  value  of  1  to 
500,000,  as  tested  in  the  treatment  of  an  animal  which 
had  received  ten  times  the  minimum  fatal  dose  of  a  two 
days'  bouillon  culture.  A  ten  per  cent,  solution  of  this 
substance  had,  therefore,  ten  times  the  value  of  Behring's 
"  normal-serum."  The  precipitated  antitoxin  was  soluble 
in  water,  and  more  readily  in  a  slightly  alkaline  solution, 
and  gave  all  the  reactions  of  an  albuminous  body.  When 
dried  in  vacuo  at  40°  C,  and  then  heated  to  102°  C,  it 
still  retained  its  antitoxic  potency. 

Ehrlich,  Kossel,  and  Wassermann  (1894)  have  made  ex- 
periments upon  goats,  which  they  found  very  suscep- 
tible to  the  action  of  the  diphtheria  poison.  Sterilized 
cultures  were  first  injected  in  gradually  increasing 
amounts  and  later  virulent  cultures.  In  this  way  they 
obtained  a  serum  which  has  a  value  sixtj'  times  that  of 


DIPHTHERIA.  159 

Behring's  "  normal  serum."  In  a  subsequent  communi- 
cation (1894)  Wassermann  gives  an  account  of  his  experi- 
ments with  the  milk  of  immunized  goats,  which  contains 
the  antitoxin  in  considerable  quantity,  and  from  which  it 
was  obtained  in  a  concentrated  form  by  the  following 
method  :  The  milk  is  obtained  in  sterilized  vessels  and  20 
c.c.  of  normal  hydrochloric  acid  is  added  to  each  litre ;  a 
sufficient  quantity  of  rennet  is  then  added  to  coagulate 
the  casein,  and  this  is  separated  from  the  liquid,  which  is 
then  shaken  up  with  chloroform  for  some  time.  The 
liquid  is  now  allowed  to  stand  in  order  that  the  butter, 
which  has  been  dissolved  by  the  chloroform,  may  sink  to 
the  bottom.  The  clear  liquid  is  then  decanted  and  the 
antitoxin  precipitated  from  it  by  means  of  ammonium 
sulphate  (thirty  to  thirty-three  per  cent.).  The  precipi- 
tate is  rapidly  dried  upon  porous  porcelain  plates,  in 
vacuo,  and  then  dissolved  in  water  in  the  proportion  of 
10  parts  for  100  of  milk  first  employed — a  concentration 
to  one-tenth.  Of  this  solution  0.125  c.c.  was  found  to 
neutralize  0.9  c.c.  of  a  toxin  which  killed  guinea-pigs, 
weighing  500  gm.  in  the  dose  of  0.1  c.c.  This  toxin  was 
an  old  bouillon  culture  of  the  diphtheria  bacillus  to  which 
0.5  per  cent,  of  carbolic  acid  had  been  added  to  preserve 
it.  In  a  communication  of  the  same  date  EhrKch  and 
Wassermann  report  that  they  have  for  some  time  had  a 
cow  immunized  to  such  a  degree  that  1  c.c.  of  its  milk 
protects  guinea-pigs  from  the  fatal  effects  of  0.9  c.c.  of 
the  above-mentioned  toxin.  The  antitoxic  value  of  the 
milk  of  an  immunized  cow  or  goat,  as  compared  with  that 
of  its  blood,  is  estimated  by  Ehrlich  and  Wassermann  as 
from  1  to  15  to  1  to  30— usually  about  1  to  20. 

Aronson,  in  testing  his  antitoxin,  uses  a  bouillon  cult- 
ure of  the  diphtheria  bacillus  two  and  one-half  mouths 


160  IMMUNITY   AND   SERUM-THERAPY. 

old,  which  he  preserves  by  the  addition  of  0.3  per  cent,  of 
trikresol.  He  fiuds  that  the  immunity  which  results 
from  injections  of  the  antitoxin  is  established  at  once ; 
that  it  is  not  accompanied  by  any  reaction  or  symptom 
of  sickness ;  and  that  it  is  of  comparatively  short  dura- 
tion. 

As  a  result  of  extended  experiments  made  at  the  Pas- 
teur Institute  in  Paris,  Koux  has  perfected  the  following 
method  for  the  production  of  an  antitoxin  suitable  for 
use  in  the  treatment  of  diphtheria  in  man.  The  horse 
has  been  found  the  most  suitable  animal  for  this  pur- 
pose, on  account  of  his  slight  susceptibility  and  the  ease 
with  which  a  high  degree  of  immunity  can  be  estab- 
lished ;  and  because  of  the  large  amount  of  blood  that 
may  be  drawn  without  injury  to  the  animal.  Roux  pre- 
pares his  toxin  by  cultivating  the  diphtheria  bacillus  in 
a  slightly  alkaline  bouillon  made  from  beef  and  contain- 
ing two  per  cent,  of  jieptone  and  0.5  per  cent,  of  sodium 
chloride.  This  medium  is  placed  in  flat-bottomed  flasks, 
and  should  not  be  more  than  half  an  inch  in  depth.  Two 
glass-tubes  pass  into  the  flask,  which  serve  for  inlet  and 
outlet  tubes  to  be  used  in  passing  a  current  of  air  over 
the  cultures.  This  is  commenced  when  the  growth  is 
fairly  started,  at  the  end  of  twenty-four  hours,  and  the 
air  should  be  moist  to  prevent  the  evaporation  of  the  cult- 
ure. In  Roux's  laboratory  a  flask  is  used  which  has  a 
tube  attached  to  one  side,  about  an  inch  from  the  bottom, 
and  which  is  known  as  a  Fernbach  flask.  A  flocculent 
deposit  falls  to  the  bottom  and  gradually  accumulates 
for  about  a  month.  This  consists  of  bacilli  which  have 
for  the  most  part  lost  their  vitality  and  are  tindergoing 
degeneration.  At  th^  end  of  thirty  days,  during  which 
time  they  are  kept  in  an  incubating  oven  at  a  tempera- 


DIPHTHERIA.  161 

ture  of  37°  C,  tlie  cultures  are  passed  through  a  Pasteur- 
Chamberland  lilter,  and  0.5  per  cent,  of  carbolic  acid  may 
be  added  in  order  to  preserve  them.  This  filtrate  is  so 
toxic  that  a  dose  of  0.1  c.c.  will  kill  a  g-uinea-pig-  weigh- 
ing 500  gm.  in  less  than  forty-eight  hours.  A  healthy 
horse  is  selected  and  receives  at  first  a  dose  of  0.5  c.c.  of 
the  filtered  culture  (or  of  the  clear  fluid  obtained  from  a 
culture  by  decautation  and  containing  0.5  per  cent,  of 
carbolic  acid).  The  dose  is  gradually  increased  at  inter- 
vals of  a  few  days,  and  is  followed  each  time  by  some 
febrile  reaction  and  tumefaction  at  the  point  of  inocula- 
tion. When  the  reaction  is  excessive,  a  little  Gram's 
solution  is  added  to  the  following  dose.  The  usual  plan 
of  treatment  is  stated  by  Kinyoun  as  follows : 

"First  day,  1  to  2  c.c.  of  j-mre  toxins,  of  which  1  to  10 
c.c.  fatal  to  a  500-gm.  guinea-pig;  eighth  day,  1  c.c; 
fourteenth  day,  Ig  c.c. ;  twentieth  daj",  2  c.c.  ;  twenty— 
eighth  da}' ,  3  c.c. ;  thirty-third  day,  5  c.5. ;  tliirt3^-eighth 
day,  8  c.c. ;  forty -third  day,  10  c.c. ;  forty-seventh  day,  20 
c.c. ;  fifty -first  day,  30  c.c.  ;  fifty-sixth  day,  50  c.c. ;  sixty- 
second  day,  50  c.c. ;  sixty-eighth  day,  60  c.c.  ;  seventy- 
fourth  day,  100  c.c. ;  eightieth  day,  250  c.c.  ;  eighty-eighth 
day,  250  c.c. 

"  When  the  first  injections  are  given  there  is  quite  a 
marked  local  and  general  reaction  to  the  poison  ;  there  is 
an  oedema  at  the  point  of  the  injection,  which  is  followed 
by  a  distinct  inflammatory  process — hard  in  the  centre 
and  soft  and  oedematous  at  its  periphery.  The  general 
reaction  is  manifested  by  a  rise  in  the  temperature,  1°  to 
2°  C,  loss  of  appetite,  and  occasionally  cramps:  The  re- 
action must  be  taken  as  the  guide  in  the  future  dosage, 
and  a  sufficient  time  must  be  allowed  to  elapse  between 
the  injections  for  the  complete  recovery  from  the  general 
and  local  effects.  As  the  quantity  of  the  toxins  is  in- 
creased the  general  effects  generally  decrease,  perhaps  a 
11 


162  IMMUNITY    AND    SERUM-TIIEnAPY. 

rise  of  a  degree  for  twenty -four  hours.  The  local  effect 
partakes  more  of  an  oedema,  and  has  the  character  of  an 
intiammation. 

"  At  a  certain  stage,  usually  after  two  months'  treat- 
ment, when  50  to  60  c.c.  can  be  injected  without  harm, 
there  is  no  general  reaction,  but  a  large  oedema  at  the 
site  of  the  injection,  which  disapjjears  within  from  tAvent}-- 
four  to  forty-eight  hours.  Toward  the  last,  even  when 
200  to  300  c.c.  are  given,  there  is  only  an  enormous  oede- 
ma, which  disappears  within  from  twelve  to  eighteen 
hours.  When  these  inordinateh'  large  quantities  can  be 
given  with  only  a  local  reaction  being  manifest,  the  horse 
has  come  well  under  the  influence,  and  the  blood  will  be 
found  to  be  rich  in  the  antitoxin. 

"  There  is  a  curious  fact  well  worth  noting  :  At  the  end 
of  the  second  month  of  the  treatment,  when  the  horse  can 
bear  as  much  as  50  to  60  c.c.  of  the  toxins  without  discom- 
fort, the  blood  will  be  found  to  contain  but  little  of  the 
antitoxin.  The  antitoxin  only  appears  after  repeated 
stimulation  of  the  cells  (?)  by  the  large  and  frequent 
doses  of  the  toxijis." 

The  subcutaneous  injections  do  not  jdeld  a  serum  as 
rich  in  the  antitoxins  as  when  the  toxins  are  injected  di- 
rectly into  the  blood-current.  '\Mien  it  is  desired  to  do 
this,  toward  the  last  of  the  treatment  the  toxins  are  in- 
jected directl}^  into  the  jugular  vein.  The  process  is 
tedious  and  requires  a  longer  time,  and  for  practical  pur- 
poses has  not  been  found  so  satisfactory  as  the  simple 
subcutaneous  injection!  The  strength  of  the  serum  is 
tested  by  using  j^oung  guinea-pigs  of  500  gms.  weight. 
One  gr.  of  the  serum  usually  will  protect  50,000  gms.  of 
guinea-pig  against  a  fresh  virulent  culture  of  the  Bacil- 
lus dipJitherlae.  This  is  the  strength  that  is  used  in  the 
hospitals.  By  the  intravenous  injections  a  serum  of  the 
protective  strength  of  1  to  100,000  can  be  obtained. 


DIPHTHERIA.  163 

When  fully  immunized  from  six  to  eight  litres  of  blood 
may  be  taken  from  a  horse  at  one  time,  but  as  a  rule  it  is 
better  not  to  take  more  than  three.  The  blood  is  drawn 
from  the  jugular  vein,  by  means  of  a  small  trocar  and 
cannula,  into  wide-mouthed  bottles  having  a  capacity  of 
21  litres  ;  these  are  jalaced  in  an  ice-chest  for  twenty- 
four  hours  to  give  time  for  the  separation  of  the  serum, 
which  is  then  transferred  to  smaller  receptacles  for  pres- 
ervation. 

The  dose  of  serum  prepared  in  this  way,  when  used  to 
protect  from  diphtheria  infection,  is  5  c.c.  for  a  child  uq- 
der  ten  years  of  age,  and  10  c.c.  for  older  children.  This 
does  not  afford  an  absolute  protection,  but  is  believed  to 
be  generally  effective,  and  in  case  of  failure  the  attack 
is  said  to  be  of  a  mild  character.  The  curative  dose  of 
Koux's  serum  is  20  c.c.  for  children,  and  30  to  40  c.c.  for 
l)atients  over  fifteen  years  of  age.  The  larger  dose  is 
divided  and  given,  at  the  same  time,  by  subcutaneous  in- 
jection in  two  places.  Antisejitic  precautions  are  taken 
in  giving  these  injections,  and  a  little  absorbent  cotton 
is  placed  over  the  puncture. 

Hesults  of  Treatment  with  Serum  of  Immune  Animals. 

We  take  the  following  statistics  from  a  recent  (Decem- 
ber, 1894)  paper  by  Woodhead  : 

"  It  may  be  pointed  out  from  statistics  that  no  fewer 
than  13,694  cases  were  notified  in  London  during  the 
year  1893.  Among  these  cases  there  was  a  mortality  of 
3,195  or  23.3  per  cent,  (see  The  Lancet  corrected  statis- 
tics). In  a  series  of  most  carefully  prepared  and  tabu- 
lated statistics,  those  of  the  Metropolitan  Asylums 
Board's    hospitals    which    receive     diphtheria    patients 


164 


IMMUNITY    AXD   SEKUM-THERAPY. 


Table  I. — Metropolitan  Asylums  Board  :  Admisgions  and   Case  Mor- 
tality, Diphtheria,  1888-93. 


Year. 

No.  of  Admissions. 

No.  of  Deaths. 

Percentage  of  Case 
Mortality. 

1888      

99 

723 

942 

1312 

2009 

2848 

46 
275 
316 
397 
583 
865 

46  4 

1889    

38  0 

1890  . 

1891 

33.5 
30  2 

1892 

29  0 

1893 

30  3 

Note. — Diptitlieria  cases  have  only  been   admitted  into  the  hospitals  since 
October  23,  18b8. 


Table  II. — Shairing  the  Mortality  at  Various  Ages  from  Diphtheria 
admiiled  into  the  Metropolitan  Asylums  Board's  Ho&pitali  in  the  Years 
1888-93. 


Ages. 

Cases  Admitted. 

Died. 

Mortality  per  cent. 

Under  1    

146 
447 
039 
826 
913 

102 
291 
388 
416 
400 

69.9 

1  to2 

65.1 

2to3 

60.7 

3  to  4 

50.4 

4  to  5 

43.8 

Totals  under  5 

2,971 

1,597 

53.0 

5  to  10 

2,402 

885 

588 

420 

209 

137 

87 

50 

27 

13 

705 
93 
23 
22 

28.6 

10  to  15 

15  to  20 

10.5 
3.9 

20  to  25 

5.2 

25  to  30 

30  to  35  

35  to  40 

13 
5 
5 
6 
4 
4 
1 
3 

4.8 
3.6 
5.7 

1 

40  to  45 

45  to  50 

50  to  55   

;-     16.8 

1 

55  to  60 

11 
6 

And  upward 

J 

Totals 

7,932 

2,481 

31.3 

DIPHTHERIA. 

Table  III. — Serum  Treatment  of  Diphtheria. 


165 


u 
1" 

Percentage 
of  Mor- 
tality. 

Percentage 

of  Previous 

Mortality. 

Rou.K,  Marti  ii,  ) 
and  Cbaillou.  \ 

Ehrligb,  Kossel,  \ 
and    Wasser-  > 
maun ) 

* Canon 

Paris 

448 

220 

15 

34 

60 

89 

233 

121 

35 

96 

128 

255 

19 

70 

65 

2 

32 

79 

109 

52 

3 

6 

19 

12 

54 

40 

5 

37 

17 

31 

4 

7 

18 

8 

9 

24.5 

23.6 

20.0 
18.0 
33.3 
12.3 
23.0 
33.1 
14.2 
38.5 
13.2 
12.1 
21.0 
10.0 
28.8 

..IT 

25.0 
11.4 

51. 7^ 

34. 7§ 
25  0 

Berlin 

*Scbubert 

1. 

*yoswinkel 

Ebrlich 

(1 

46.5 

flvQssel 

*Korte 

.' 

'< 

53  8 

Bokai 

Heubner ^  . . 

Katz 

Aronsou 

Budapest 

Beriin  ".'.*.'.".;!'.; 

53.8 
62.5 
38.911 
32  5-41  7 

Ranke  and  Oertel. 

The  Lancet  

Weibgen 

Muchleck 

Dr.  White 

Cases  reported  in 
British  Medical 

Austrian  Riviera 

Berlin 

Pbiladelphia 

i  WillVl  Parker ) 

-  H  0  s  p  i  t  a  1 ,  [- 

(  New  York. . .  ) 

The  Lancet  and  ] 

Journal f 

40.0 
42.7 

*  Canon,  Schubert,  and  Voswinkel's  statistics,  and  probably  also  the  bulk  of 
Korte's,  are  really  included  in  the  Ehrlich,  Kossel,  and  Wassermann's  table. 

+  Kossel's  statistics  appear  to  be  simply  an  extension  of  those  given  by  Ehr- 
lich, Kossel,  and  Wassermann. 

I  Mortalitj' in  Trousseau  Hospital  at  same  period  without  use  of  serum  63.2 
per  cent. 

§  Based  on  previous  seven  years'  statistics. 

1  Based  on  three  years'  statistics.     Last  year's  mortality  was  41.7  per  cent. 

1[  Tvyo  not  treated  died. 


(Table  I.)  we  find  that  during-  the  year  1893  the  case  mor- 
tality among-  eases  of  diphtheria  admitted  to  the  Avards  of 
these  hospitals  was  a  little  OAer  thirt}'  per  cent. — under 
conditions  which  we  may  say  are  practically  unknown  in 
France  and  in  most  parts  of  Germany  and  Austria.  This 
is  sufficiently  startling-,  but  if  we  examine  earlier  statis- 


166  IMMUNITY   AND   SERUM-THERAPY. 

tics  we  find  that  duriner  tlie  years  1888-90  the  case  mor- 
tality, as  indicated  by  tiie  admissions  and  deaths,  "svas 
much  higher.  On  reference  to  Table  I.  it  will  be  seen 
that  since  the  year  1888  the  case  death-rate  has  fallen 
from  4G.4  to  30.3  per  cent.,  althoug-h  the  number  of  cases 
of  diphtheria  treated  has  risen  enormously.  The  mortal- 
ity from  diphtheria  in  London  during-  the  same  period 
has  also  risen  very  considerably.  The  case  mortality 
at  different  ages  is  brought  out  on  reference  to  Table  II. 
Comparing  these  results  of  treatment  in  our  hospitals 
with  those  obtained  in  the  Paris  and  Berlin  hospitals  (see 
Table  III.),  the  records  of  which  are  published,. it  is  at 
once  seen  how  much  more  favorable  are  the  results  .ob- 
tained in  the  hospitals  of  the  Metropolitan  As^dums 
Board.  It  will  be  noted,  however,  that  the  figures  apply- 
ing to  the  earlier  years  represented  a  case  mortality  al- 
most as  high  as  obtained  in  the  French  and  German  hos- 
pitals immediately  before  the  antitoxic-serum  treatment 
was  introduced.  The  mortality  in  hospitals  must  always 
be  greater  than  in  private  practice  from  the  fact  that  so 
many  of  the  cases  are  so  serious ;  some  of  the  patients, 
indeed,  are  actually  moribund  when  aelmitted." 

Fischer  (January,  1895)  reports  34  cases — 30  malignant 
and  4  mild — treated  with  a  mortality  of  two  =  5.8  per 
cent.     He  says: 

"  These  cases  were  not  selected,  for  some  were  poorly 
nourished,  some  in  excellent  vitality,  with  careful  nurs- 
ing and  good  hj' giene.  The  main  point  was  to  appl}'  the 
antitoxin  as  early  as  possible,  and  counteract  the  septic 
matter  absorbed,  and  thereby  avoid  complications,  be- 
sides using  the  local  treatment  of  swabbing  the  throat 
with  a  1  to  2,000'  bichloride  of  mercury  solution,  using  a 
fresh  swab  for  each  application,  and  burning  the  same 
immediately  after  using  it. 

"The  technique  of  injection  is  simple.  Having  prop- 
erly sterilized  the  syringe  by  boiling  and  using  0.5  per 


DIPHTHERIA.  167 

cent,  tricresol,  I  commence  by  injecting-  10  c.c.  in  mild 
cases,  and  20  c.c.  in  malignant  cases,  by  i^incliing  a  fold 
of  the  skin  in  tlie  intrascapular  region,  and  allowing  the 
serum  to  be  slowly  injected.  I  believe  it  proper,  however, 
to  have  a  syringe  of  suitable  size  and  inject  the  required 
amount,  rather  than  inject  several  places.  The  calibre  of 
the  latter  must  necessarily  be  quite  large,  owing  to  the 
thickness  of  the  serum,  which  is  at  times  rather  mucilag- 
inous. It  is  proper  to  note  all  differences  and  effects  on 
the  false  membrane  and  the  swelling  of  the  glands,  the 
behavior  of  the  temi:>erature,  the  condition  of  the  urine, 
the  effect  on  the  heart,  and  especially  the  pulse. 

"  There  should  be  no  hesitation  in  injecting  on  the  sec- 
ond day,  and,  if  no  effect  is  seen,  repeating  the  injection 
on  the  third  day,  as  there  is  absolutely  no  risk  from  the 
injection.  It  is  a  perfectly  safe  remedy,  and  shows  no 
immediate  reaction.  It  differs  from  tuberculin  and  vac- 
cine in  that  it  causes  no  reaction.  A  case  of  antitoxin 
treatment  will  show  no  symptoms  directly  attributable  to 
the  remedy,  unless  it  be  in  some  cases  of  urticaria.  The 
temperature  does  not  fall  by  crisis,  but  by  lysis,  with  an- 
titoxin treatment.  Massage  of  the  serum  after  the  injec- 
tion should  not  be  practised,  according  to  Heubner, 
Aronson,  Baginsky,  and  others." 


The  efficacy  of  the  antitoxin  treatment  has  been  com- 
batted  in  Berlin  by  Hansemann  and  others.  The  oppo- 
nents of  the  treatment  point  to  a  considerable  number  of 
recorded  cases  in  which  relapses  have  occurred  after  ap- 
parent recovery  following  treatment  with  the  antiseptic 
serum  ;  also  to  the  occurrence  of  diphtheria  in  children 
who  had  been  treated  with  immunizing  doses  of  the 
serum  ;  and  to  the  failure  to  cure  in  a  considerable  num- 
ber of  cases  notwithstanding  the  administration  of  large 
doses  of  the  antitoxic  serum.  All  of  these  facts  must  be 
admitted,  but  the  experimental  (on  animals)  and  clinical 


168  IMMIXITY    AND    SEIIUM-THEIIAPY. 

evidence  heretofore  submitted  appears  to  establish  the 
value  of  the  treatment  when  applied  before  the  disease 
has  i^rogressed  too  far.  It  must  be  remembered  that  the 
antitoxin  has  no  power  to  destroy  the  diphtheria  bacilli, 
or  to  relieve  the  suffocation  resulting  from  obstruction  of 
the  larynx,  or  to  cure  an  acute  parenchymatous  nephritis 
due  to  the  action  of  the  deadly  toxin  elaborated  by  the 
Klebs-Loffler  bacillus. 

Saltmann  (1895)  reports  a  mortality  of  39.8  per  cent,  in 
71  cas3S  treated  in  the  children's  hospital  at  Leipsic  be- 
fore the  use  of  the  antitoxin.  The  report  relates  to  cases 
treated  from  April  1,  to  December  31,  1894.  During  the 
last  five  months  of  this  period  122  cases  were  treated  and 
most  of  them  received  the  antitoxin.  The  mortality  was 
18  per  cent. 

In  an  article  contributed  to  McClure^s  Magazine  for 
March,  1895,  Dr.  Hermann  Big-g-s,  of  New  York,  saj-s  : 
"  That  prepared  in  this  country  under  the  supervision  of 
the  New  York  City  Health  Department  has,  at  the  time 
of  writing,  been  already  employed  in  more  than  one  hun- 
dred and  fifty  cases,  and  the  mortalit}^  in  the  cases  thus 
treated  has  been  about  twelve  per  cent." 

Klebs  has  proposed  to  treat  diphtheria  with  a  prepa- 
ration obtained  from  cultures  of  the  diphtheria  bacillus 
which  he  calls  antidiptherin.  The  clinical  experiments 
made  by  Yulpius  and  by  Zappert  show  that  the  treat- 
ment has  no  value  and  it  has  been  abandoned. 

BIBLIOGRAPHY. 

Abel,  R.  :  Ueber  die  Scluitzkraft  dcs  Blutserums  von  Diplitberierekon- 
valescenten,  etc.     Dtscbe  Med.  AVcbsolir. ,  1894.  p.  899. 

Aronson  :  Experimeiitale  Untcrsucbunsren  iiljcr  Dipbtberie  und  die  im- 
munisierende  Substanzdes  Blutserums.  Bed.  klin.  Wcbscbr.,  1893, 
Nos.  25  and  26  ;  Centialbl.  f.  Bakteriol.,  XIV.,  p.  260. 


DIPnTHERIA.  169 

Aronson  :  Ueber  Diplithcrieantitoxin  losungzii  Immunisierungszweck- 

en.     Dtscb.  med.  Wchscbv.,  1894,  p.  431. 

Zur  Diphtberiebeihiugsfiage.   Dtscb.  Med.  Wcbscbr.,  1894,  p.  384. 

Weitere  Untersucbungeu  iibcr  Dipbtberie  und  das  Dipbtberie- 

autitoxiti.     Berl.  kUn.  Wocbeuscbr.,  1894,  pp.  356,  42.1,  453.     Abst. 

in  Ceutralbl.,  XV.,  926. 
Meine   Stelluug  in   der   Dipbtberieantitoxiufrage.      Berl.    klin. 

Wcbscbr.,  1894.  No.  47,  p.  1077. 

Iiiimunisieruugs- uud  Heilversucbe  bei  der  Diplitberie  mittelst 


Antitoxin.    Wien  med.  Wcbscbr.,  1894,  Nos.  46-48,  pp.  1953,  2001, 

2046. 
Bebring  :  Untersucbnngen  iiber  das  Zustandekommen  der  Dipbtberie- 

immunitiit  bei  Tbieren.     Deutscbe  Med.  Wcbscbr.,  1890,  No.  50. 
Die  Blutserumtberapie  bei  Dipbtberie  und  Tetanus.    Zeitscbr.  f. 

Hygiene,  XII.,  1,  1883. 
Zur     Bebandluug    der    Dipbtlierie    mit     Dipbtberiebeilserum. 

Dtscbe.  med.  Wcbscbr..  1893,  pp.  543,  594,  623,  637. 
Weitere   Bemerkungen   zur   Dipblberiebeilungst'rage.      Dtscbe. 

Med.  Wcbscbr.,  1894,  No.  32,  p.  645. 
Zur   Diplitlierieimmunisierungsfrage.     Dtscbe.   med.    Wcbscbr. 

1894,  No.  46,  p.  865. 
Die  Blutserumtberapie  zur  Dipbtberiebebandlung  des  Menscben. 


Berl.  klin.  Wcbscbr.,  1894,  p.  827. 
Bebring  und  Kitasato  :  Ueber  das  Zustandekommen  der  Dipbtberieim- 

munitiit  und  der  Tetanusimmunitat  bei  Tbieren.     Deutscbe  med. 

Wcbscbr.,  1890,  No.  49. 
Bebring  und  Wernicke  :  Ueber  Immunisierung  und  Heilung  von  Ver- 

sucbstbieren  bei  der  Dipbtberie.     Zeitscbr.  f.  Hygiene,  XII.,  10, 

1892. 
Bebring,  Boer,  und  Kossel  :  Zur  Bebandlung  dipbtberiekranker  Men- 
scben  mit  Dipbtberiebeilserum.     Dtscbe.    med.    Wcbscbr.,   1893, 

389,  415. 
Bebring  und  Boer  :  Ueber  die  quantitative  Bestimmung  von  Dipbtberie- 

antitoxin.    Dtscbe.  med.  Wcbscbr..  1894,  No.  21. 
Bokai,   J.  :   Mein   auf  der  Dipbtberieabtbeilung    des    Stefaniekinder- 

spitals  mit  dem  Behring'scben  Heilserum  bisber  erzielten  Resultnte. 

Pester  Med.  Cbirug.  Presse.,  1894,  Nos.  44  and  45,  pp.  1120,  1143. 
Brieger   und   Frankel  :    Untersucbungen    iiber    Bakteriengifte.     Berl. 

klin.  Wcbscbr.,  1890,  Nos.  11  and  12. 
Ueber    Immuni-sierungsversucbe    bei    Dipbtberie.     Berl.    klin. 

Wcbscbr.,  1890,  No.  49. 
Canon :     Zur    Dipbtberiebebandlung    mit    Heilserum.      Dtscli.    med. 

Wcbscbr.,  1894,  Nos.  23  and  24. 
Ebrllcli  und  Kossel :  Ueber  die  Anwendung  des  Diphtberieantitoxins. 

Zeitscbr.  fiir  Hygiene,  Bd.  XVII.,  1894,  p.  486. 


170  IMMUNITY   AND   SERUM-THERAPY. 

Eliiiicb,  P.,  u.  Wassermann,  A.  :  Ueberdie  Gewinimng  der  Diplitherie- 
autiloxineaus  Blutscrum  uiid  Milch  iminunisierter  Tliiore.  Ztsclir. 
fiir  Hygiene,  1894,  Bd.  XVIII.,  p.  238. 

EUrlich,  Ivossel,  und  "Wassermann  :  Ueber  Gewinnung  uud  Verwen- 
dung  des  Diphlberiebeilserums.  Dtscb.  med.  Wocbenscbr.,  1894, 
Xo.^G. 

Hausemauu  :  Mittheilungen  iiber  Diplitberie  und  das  Dipbtheiie-Heil- 
serum.     Berl.  klin.  Wcbscbr.,  1894.  p.  595. 

Hilbert,  P.  :  Die  Resultate  der  in  der  Kgl.  mediziniscbeu  Uuiversitats 
Poliklinik  zu  Kouigsberg  ausgefiibrten  Scbutz-und  Heilimpfungen 
bei  Dipbtherie.     Berl.  klin.  Wcbscbr.,  1894,  Xo.  48,  p.  1084. 

Kiuyon  :  The  Seruuitberapy  of  Dipbtberia.  Abst.  of  Sanitary  lie- 
ports,  vol.  ix.,  Xo.  51,  1894. 

Klebs :  Ein  Beitrag  zur  Antidipbtbcrin  Bebandlung.  Dtscb.  med. 
Wcbscbr.,  1894,  Xo.  18. 

Klemensiewicz,  R.,  und  Escbericb,  Tb.  :  Ueber  einen  Scbutzkorper  im 
Blute  von  Dipbtberie  gebeilten  Mensclieu.  Centralbl.  f.  Bakteriol., 
XIII.,  153,  1893. 

Korte,  W.:  Bericbt  iiber  die  Bebandhing  von  121  Dipbtberiekranken 
mit  Bebring'scben  Ileilserum  im  stadtiscben  Krankenbaus  am  Ur- 
ban.    Berl.  klin.  W(discbr.,  1894,  Xo.  46,  p.  1039. 

Kossel,  H.  :  Ueberdie  Bebaudhing  der  Dipbtberie  des  Menscben  mit 
Diptberiebeilserum.     Zeitscbr.  fur  Hyg.,  XVII.,  489,  1894. 

Koudrevetsky,  B.  :  Kecbercbes  e.xperimeutales  sur  I'immunisation 
contre  la  dipbtberie.     Arcb.  de  ]VIed.  experiui.,  1893,  p.  620. 

Kuprianow,  J.  :  Experiinentelle  Beitrage  zur  Frage  der  Immunilat  bei 
Dipbtberie.     Centralbl.  f.  Bakteriol.,  XVI..  415,  1894. 

Lubinski,  W.  :  Ueber  eine  Xachwirkung  des  Antitoxins  bei  Beband- 
lung der  Dipbtberie.     Dtscbe.  med.  Wcbscbr., 1894,  Xo.  45,  p.  857. 

Roux  et  Yersin  :  Contribution  a  I'etude  de  la  dipbtberie.  Ann.  de 
rinst.  Pasteur,  t.  ii.,  1888,  p.  629. 

Roux,  E.,  Martin,  L.,  et  Cbaillou  :  Trois  cents  cas  de  dipbtberie  traites 
par  le  serum  antidipbtberique.  Ann.  de  I'lnst.  Pasteur,  1898,  p. 
640. 

Risso,  A.:  Ueber  die  Immunisation  von  Tbieren  gegen  Dipbtberitis 
und  iiber  die  Serumiberapie.  Abst.  in  Centralbl.  fur  Bakteriol., 
XVI. ,  244,  1894. 

vSaUmann  :  Die  Serum  Bebandlung  der  Dipbtberie.  Deutsche  med. 
Wcbscbr.,  1895,  p.  53. 

Schubert,  E. :  Ueber  die  mit  dem  Bebring-Elirlicb'scben  Diptberiebeil- 
.serum  gemachten  Erfabrungen.  Dtscbe.  med.  Wcljscbr.,  1894,  p. 
476. 

Smirnow  :  Ueber  die  Bebandlung  der  Dipbtberie  mit  Antitoxinen,  die 
ohne  Yermittclung  des  thieriscben  Organismus  darstellbar  sind. 
Berl.  klin.  Wcbnscbr.,  1894,  p.  683  ;  Centralbl.,  XVI.,  546. 


DIPHTHERIA.  171 

Strauss  :  Sur  la  serumtherapie.     Rapport  d'uue  commission.     Bull,  de 

I'Acad.  de  Med.,  1894,  No.  43,  p.  329. 
Uschinski,  N. :  Ueher  die  chemische  Beschaffenheit  des  Diplitherie-nnd 

Choleragiftes.     Bolnitscli.  Gaz.  Botkiua,  1893,  pp.  437,  453,  485. 

(Russiau.) 
Vo.swinckel :  Resultate  der  Heilserumtlierapie  bei  Diplitlierie.     Abst. 

in  Ceutralbl.  f.  Bakteriol.,  XVI.,  548,  1894. 
Vulpius  :    Kritisclie  Bemerkungen  und  praktische    Erfalmngen  iiber 

das  Antidipbtherin  Klebs.     Dtscb.  med.  Wcbscbr.  1894,  No.  6. 
Wassermann,  A.:  Ueber  Konsentrieruiig  der  Dipbtberieantitoxin   aus 

der  Milcb  immunisierter  Tbiere.     Ztscbr.    f.    Hygiene,  1894,  Bd. 

XVIII.,  p.  235. 
Wernicke  :  Ein  experimeuteller  Beitrag  zur  Kenutniss  des  Loffler'scben 

Dipbtberiebacillus  und  zur  "  Blutserumtberapie."     Arcb.  f.  Hyg., 

1893,  p.  193. 
Beitrag  zur  Kenntniss  des  Loifler'scben   Dipbtberiebacillus  und 

zur  Blutserumtberapie  bei   Dipbtberie.     Abst.    in   Centralbl.  I'iir 

Bakteriol.,  XV.,  898,  1894. 
Woodbead,  G.  S.  :  Tbe  Diagnosis  and  Antitoxic  Serum  Treatment  of 

Dipbtberia.     Tbe  Lancet,  London,  December,  15,  1894,  p,  1409. 
Zappert,  J.;  Ueber  die  Heilwirkung  des  Antidipbtberin  (Klebs).  Wieu 

med.  Wcbscbr.,  1894,  pp.  550,  616,  666,  708,  756. 
Zimmer  E. :  Untersucbungen  iiber  das  Zustandekommeu  der  Dipbtberie- 

immuuitat.     Dtscbe.  med.  Wcbscbr.,  1893,  No.  16. 


V. 

FOOT-AND-MOUTH   DISEASE. 

This  is  an  infectious  disease  of  cattle,  sheep,  goats,  and 
swine,  the  etiology  of  which,  so  far  as  the  specific  infec- 
tious agent  is  concerned,  has  not  been  determined. 
Schottelius  has  (1892)  described  a  microorganism  found 
by  him  in  the  contents  of  the  vesicles  of  foot-and-mouth 
disease  which  seems  to  be  peculiar  to  this  disease,  but 
he  was  not  able  to  demonstrate  its  etiological  relation  by 
inoculation  experiments.  The  microorganism  referred 
to  is  a  streptococcus  so  far  as  its  morphology  is  con- 
cerned, but  differs  from  the  previously  known  strepto- 
cocci in  being  extremely  motile. 

The  extent  to  which  the  disease  in  question  prevails  in 
some  parts  of  Europe  is  shown  by  the  statistics  for  1891 
of  the  prevalence  of  this  disease  in  Germany.  According 
to  the  Reichsseuchenbericht  it  prevailed  most  exten- 
sively in  the  southern  portion  of  Germany.  The  total 
number  of  infected  farms  was  47,865  ;  the  total  number  of 
infected  cattle  was  394,640 ;  of  sheep,  240,904 ;  of  goats, 
3,378  ;  of  swnie,  182,208.  Behla  (1892)  has  made  inocula- 
tion experiments  with  the  filtered  saliva  of  infected  cattle 
to  which  he  added  one  to  two  per  cent,  of  carbolic  acid, 
and  claims  to  have  produced  immunity  in  young  pigs 
and  lambs.  The  duration  of  immunity  is  not,  however, 
very  long  even  in  animals  which  have  recovered  from  an 
attack  of  the  disease — said  to  be  from  six  months  to  three 


FOOT-AND-MOUTH   DISEASE.  173 

years — and  a  practical  method  of  restricting"  the  disease 
by  means  of  protective  inoculations  has  not  as  yet  been 
introduced. 

BIBLIOGEAPHY. 

Behla,  R.  :    Zur  Schutzimpfung  bei   Klauen-  uud  Maulseuchc.     Beil. 

tierarztl.  Wclisclir.,  1892,  No.  49. 
Sanfelice,  F.  :    Ueber  einen  Befuiid  an  von  Maul-  und  Klauenseiiclie 

befalleuen  Tieren.     Centralbl.  f.  Bakteriol.,  1894.  No.  23,  p.  896. 
Schiitz  :  Impfversuche  zum  Schutze  gegeu  die  Maul-  und  Klauenseuche. 

Arch,  fiir  Wissenscb.  u.  Prakt.  Tierbeilk.  1894,  No.  1,  p.  1. 
Scbottelius :    Ueber  einen   bakteriologiscben   Befund   bei    Maul,    und 

Kll,uenseuche.  Centralbl.,  f.  Bakteriol.     Bd.,  XI.,  1893,  p.  566. 


VI. 

Glanders. 

The  etiological  relation  of  the  bacillus  discovered  by 
Loffler  and  Schiitz,  in  1882,  to  glanders  is  well  estab- 
lished. Experiments  by  the  bacteriologists  named,  and 
frequently  repeated  by  others,  show  that  pure  cultiires  of 
this  bacillus  injected  into  horses,  asses,  and  other  suscep- 
tible animals,  produce  genuine  glanders.  The  field- 
mouse  and  the  guinea-pig  are  especially  susceptible  to 
infection  by  experimental  inoculations  ;  the  cat  and  the 
goat  may  also  be  infected ;  rabbits,  sheep,  and  dogs  are 
but  slightly  susceptible,  and  swine,  cattle,  white  mice, 
and  house-mice  are  immune. 

The  toxic  substances  produced  in  cultures  of  the  glan- 
ders bacillus  when  concentrated  in  the  form  of  a  glj^ceriu 
extract  constitute  the  so-called  mallein,  which  has  been 
extensively  used  in  the  diagnosis  of  glanders  in  horses. 
As  is  the  case  when  animals  infected  with  tuberculosis 
are  inoculated  with  tuberculin,  animals  infected  with 
glanders  have  a  decided  rise  of  temperature  after  receiv- 
ing a  sufficient  dose  of  mallein  beneath  the  skin. 

Babes  (1892)  reports  that  the  toxic  substance  in  cult- 
ures of  the  glanders  bacillus  may  be  obtained  by  precipi- 
tation with  alcohol ;  and  that  mallein  obtained  from 
filtered  cultures  to  which  glycerin  has  been  added,  or  the 
alcoholic  precipitate,  may  be  successfully  used  for  pro- 
tecting susceptible  animals  against  glanders  infection  or 


GLANDERS.  175 

for  curing-  the  disease  after  infection.  He  has  demon- 
strated the  therapeutic  value  upon  g-uiuea-pigs  and 
upon  two  horses  which  are  said  to  have  been  cured  of 
chronic  glanders.  When  large  and  repeated  doses  are 
injected  into  healthy  animals  they  produce  nephritis  and 
general  marasmus.  The  action  upon  horses  infected 
with  glanders  is  very  marked  and  small  doses  may  even 
cause  death. 

Kresling  (1892)  recommends  potato  cultures  as  prefer- 
able to  bouillon  cultures  for  the  preparation  of  mallein. 
The  potatoes  are  to  be  washed,  before  sterilization,  in  a 
five  per  cent,  bicarbonate  of  soda  solution,  "until  the 
wash-water  remains  clear."  They  are  then  cooked  for  an 
hour  and  twenty  minutes.  After  planting  uj)on  the  sur- 
face glanders  bacilli  from  a  previous  culture  they  are 
placed  in  an  incubator  at  36°  to  36.5°  C,  with  provision  to 
l^revent  them  from  becoming  dry.  At  the  end  of  two 
weeks  the  growth  is  removed  with  a  platinum  spatula 
and  added  to  nine  parts  of  water,  in  which  it  is  well 
mixed  by  rubbing.  It  is  then  allowed  to  stand  for 
twenty-four  hours,  after  which  it  is  sterilized  for  fifteen 
minutes  at  110''  C.  (a  lower  temperature  would  no  doubt 
answer  quite  as  well).  After  cooling  it  is  passed  through 
a  Chamberlain  filter  by  means  of  a  pressure  of  six  atmos- 
pheres. The  filtrate  is  then  carefully  evaj)orated  over  a 
water-bath  to  one-  fourth  its  volume,  and  to  this  concen- 
trated extract  glycerin  is  added  in  the  proportion  of  one 
part  to  two.  The  mixture  is  again  sterilized  in  the  auto- 
clave at  110°  C.  When  injected  into  healthy  horses  in 
the  dose  of  2  c.c.  this  mallein  does  not  cause  an  elevation 
of  temperature  exceeding  0.5°  to  0.8"  C.  But  1  c.c.  in- 
jected into  a  horse  having  glanders  causes  its  tempera- 
ture to  mount  to  40"  C,  and  at  the  point  of  inoculation  a 


176  IMMUNITY   AND   SEIiUM-TIIERAPY 

considerable  swelling-  is  developed,  which  lasts  from  four 
to  six  days — in  healthy  horses  a  swelling-  the  size  of  a 
man's  list  is  developed  at  the  point  of  inoculation,  which 
disappears  within  twenty-four  hours. 

In  Pasteur's  laboratory,  according-  to  Nocard  (1892), 
mallein  is  prepared  as  follows :  The  g-landers  bacillus  is 
first  made  so  virulent  by  successive  inoculations  in  sus- 
ceptible animals  that  it  will  kill  a  rabbit  or  a  white 
mouse  in  a  few  hours.  This  virulent  bacillus  is  culti- 
vated in  g-lycerin-pepton-flesh-infusion  (five  per  cent,  of 
glycerin  and  five  per  cent,  of  pei3ton).  The  cultures  are 
kept  in  the  inciibating-  oven  for  four  weeks  at  a  tempera- 
ture of  31°  C,  and  then  sterilized  in  the  autoclave  at  110° 
C.  They  are  then  filtered  throug-h  paper  and  evaporated, 
in  vacuo,  over  sulphuric  acid,  at  a  low  temperature,  to 
one-tenth  of  the  original  volume.  The  result  is  a  sirup- 
like, dark  brown,  strong-smelling-  liquid,  which  is  about 
one-half  glycerin.  This  can  be  preserved  in  a  cool  and 
dark  place  for  a  long  time.  When  it  is  to  be  used  nine 
parts  of  a  0.5  per  cent,  solution  of  carbolic  acid  are  added 
to  one  part  of  the  glycerin  extract.  The  concentrated 
extract,  when  injected  into  a  healthy  horse  in  the  dose  of 
0.5  to  1  c.c,  causes  a  local  swelling  which  disappears 
after  two  or  three  days.  The  temperature  of  the  body  is 
elevated  from  1.5°  to  2°  C.  as  a  result  of  the  injection,  and 
there  is  chilliness,  loss  of  appetite,  and  debility.  When 
the  diluted  mallein  is  injected  in  healthy  animals  in  the 
dose  of  2.5  c.c.  no  reaction  occurs.  On  the  other  hand, 
this  dose  causes  an  intense  febrile  reaction  in  horses 
with  glanders.  There  is  a  chill  followed  by  an  elevation 
of  temperature  amounting  to  2°  to  3°  C,  accompanied  by 
dyspnoea  and  great  debility  ;  in  some  cases  the  animal 
died  as  a  result  of  the  inoculation. 


GLANDERS.  177 

For  the  preparation  of  the  active  substance  in  a  dry 
condition,  Foth  gives  the  following  directions  :  The  cult- 
ures are  evaporated  at  a  temperature  not  exceeding-  80° 
C.  to  one-tenth  of  their  volume,  and  filtered.  The  clear 
and  thick,  dark  brown  liquid  is  then  slowly  dropped  into 
absolute  alcohol  (25  to  30  parts)  with  constant  stirring. 
A  flaky,  white  precipitate  is  thrown  down,  and  accumu- 
lates as  a  i^ale  yellow  mass  upon  the  sides  and  bottom  of 
the  vessel.  After  standing  for  twenty-four  hours  the 
alcohol  is  carefully  drawn  off  and  the  i^recipitate  washed 
with  absolute  alcohol.  This  is  to  be  carefully  done,  and 
to  avoid  loss  will  require  several  days.  The  precipitate 
is  then  placed  upon  a  thick  paper  filter  and  thoroughly 
washed  by  drawing  alcohol  through  it  by  means  of  an 
exhaustion  apparatus,  after  which  the  purified  precipi- 
tate is  collected  and  dried  with  care  at  a  low  temperature 
— best  in  a  vacuum  over  sulphuric  acid.  A  spongy,  crum- 
bling mass  is  thus  obtained,  which  is  easily  crushed  to 
form  an  extremely  light  white  powder.  This  is  readily 
soluble  in  water.  It  is  not  at  all  hygroscopic,  and  can  be 
preserved  in  a  dry  condition  without  difficulty.  The  dose 
for  a  horse  is  0.1  gm. 

De  Schweinitz  and  Kilborne,  in  a  paper  published  in 
November,  1892,  state  that  in  December,  1890,  they 

"  Extracted  from  culture  liquids  of  the  bacillus  mal- 
leus an  albumose  which  appeared  to  be  the  active  princi- 
ple in  these  cultures.  At  that  time  a  preliminary  exper- 
iment was  conducted  to  see  if  this  substance  could  be 
used  to  make  guinea-pigs  immune  to  the  disease — glan- 
ders. The  result  was  that  out  of  a  set  of  five,  three  vac- 
cinated and  two  checks,  only  one,  a  vaccinated  animal, 
recovered  from  an  inoculation  of  a  glanders  culture. 
This  experiment  has  since  been  repeated  with  sets  of  ten 
12 


178  IMMUNITY   AND   SERUM-THERAPY. 

and  twelve  guinea-pigs  each,  with,  at  present  writing, 
only  negative  results.  A  note  of  this  work  was  published 
in  the  '  Annual  lieport  of  the  Department  of  Agriculture 
for  1891.'  The  albumose  was  best  obtained  from  the  cult- 
ures, after  the  removal  of  the  germ,  by  means  of  a  Pasteur 
filter,  by  precipitation  with  absolute  alcohol,  resolution 
in  water,  and  reprecipitation." 

Babes  (1892)  claims  to  have  succeeded  in  immunizing 
guinea-pigs  against  glanders  by  means  of  the  toxic  sub- 
stances contained  in  cultures  of  the  bacillus. 

In  a  recent  paper  (1891)  Foth  has  reported  the  results 
of  extended  experiments  which  have  been  made  with  his 
"  Malleinum  siccum  "  in  Austro-Hungary.  These  results 
are  stated  as  follows  : 

The  experiments  were  for  the  most  part  made  by  Pro- 
fessor Schiudelka,  of  Vienna.  The  tests  were  made  with 
doses  ranging  from  0.01  gm.  to  0.02  gm.  The  number  of 
horses  treated,  for  diagnostic  purposes,  was  455  ;  of  these 
147  were  examined  post-mortem.  In  general  the  infected 
horses  reacted  and  the  others  did  not.  A  reaction  of  2°  C. 
and  upward,  running  a  typical  course,  was  evidence  that 
the  animal  was  infected,  and  such  animals  were  killed 
and  carefully  examined  by  autopsy. 

A  reaction  of  1.3°  to  1.9°  C,  running  a  typical  course, 
was  taken  as  evidence  that  the  animal  was  probably  in- 
fected, and  called  for  its  isolation  and  a  subsequent  in- 
oculation after  an  interval  of  four  weeks. 

A  reaction  of  less  than  1.2°  C,  or  an  atj'pical  course  of 
the  febrile  reaction,  was  taken  as  evidence  of  non-infection. 

The  typical  febrile  reaction  consisted  in  a  rapid  or 
gradual  elevation,  according  to  the  dose,  then  a  fall  of 
some  tenths  of  a  degree,  a  subsequent  elevation  to  the 
highest  previously  reached  point  or  above,  and  a  gradual 


GLANDERS.  179 

fall  to  the  normal.  Tlie  at3'pical  reaction,  which  some- 
times occurs  in  healthy  animals,  consists  in  an  early  and 
rapid  elevation  followed  by  an  equally  rapid  fall  to  the 
normal.  To  properly  distinguish  the  typical  temper- 
ature curve,  upon  which  the  diagnosis  depends,  hourly 
observations  are  considered  necessary. 

Schiitz  (1894^,  as  a  result  of  his  experiments  on  fifty- 
four  horses,  arrives  at  the  conclusion  that  mallein  may 
give  rise  to  the  so-called  "  typical  reaction  "  in  horses 
which  are  not  infected  with  glanders. 

Hutyra  and  Preiz  (1894),  as  a  result  of  their  extended 
researches,  arrive  at  the  conclusion  that  the  use  of  mallein 
constitutes  the  most  important  means  for  the  early  diag- 
nosis of  glanders  in  horses.  They  conclude  that  a  temper- 
ature of  39.4°  C.  may  be  accepted  as  a  safe  positive  mal- 
lein reaction.  According  to  them  the  reaction  commences 
from  four  to  six  hours  after  the  injection,  and  reaches  its 
maximum  in  from  eight  to  fourteen  hours — rarely  in  six- 
teen to  twenty  hours.  The  return  to  the  normal  occurs 
in  from  twenty-four  to  thii"ty-six  hours.  The  authors  last 
named  give  the  following  directions  for  the  preparation 
of  mallein  :  The  virulence  of  the  glanders  bacillus  is  first 
increased  by  passing  it  through  a  series  of  guinea-pigs. 
Cultures  are  then  made  upon  sterilized  potato.  When 
the  culture  and  potato  have  become  quite  dry  and  dark 
colored  they  are  collected  in  a  glass  dish  and  covered 
with  a  liquid  consisting  of  equal  parts  of  glycerin  and  dis- 
tilled water,  containing  3  to  5  parts  per  thousand  of  mer- 
curic chloride.  After  standing  for  from  ten  to  fourteen 
days  in  an  incubating  oven  at  37.5°  C,  the  liquid  is  fil- 
tered through  paper  and  sterilized  for  an  hour  in  a  steam- 
sterilizer.  This  liquid  remains  sterile  on  account  of  the 
presence  of  mercuric  chloride,  and  may  be  preserved  a 


180  IMMUNITY    AND   SERUM-THERAPY. 

long  time  without  losing  its  activity.  The  dose  is  from 
0.3  to  0.5  c.c,  which  is  diluted  to  3  c.c.  with  carbolic  acid 
water  (0.5  per  cent,  solution).  The  diluted  solution  may 
also  be  kept  a  long  time  without  losing  its  activity. 

Bonome  and  Yivaldi  (1892)  have  tested  the  action  of 
mallein  obtained  by  precipitation  with  alcohol  upon 
various  animals.  Guinea-pig^  were  found  to  resist  com- 
paratively large  doses  (10  to  15  milligr.),  while  rabbits 
and  cats  were  more  sensitive  to  the  toxic  action.  In 
guinea-pigs  and  rabbits  infected  with  glanders  bacilli 
very  small  doses  had  a  favorable  influence  upon  the  prog- 
ress of  the  infection,  and  in  healthy  guinea-pigs  a  cer- 
tain degree  of  immunity  was  induced  by  the  repeated  in- 
jection of  small  doses. 

In  a  subsequent  paper  (1894)  Bonome  reports  that  he 
has  had  favorable  results  in  the  treatment  of  chronic 
glanders  in  man  by  doses  of  y^  to  ^V  c.c  The  first  dose 
is  said  to  have  caused  an  elevation  of  temperature,  head- 
ache, polyuria,  etc.,  but  upon  repeating  the  dose  after 
two  or  three  daj^s  a  decided  improvement  of  the  general 
symptoms  followed. 

Chenot  and  Picq  (1892)  claim  to  have  cured  glanders 
in  guinea-pigs  by  injections  of  blood-serum  from  the  ox, 
which  animal  has  an  immunity  from  the  disease.  They 
also  state  that  the  blood-serum  of  the  ox  is  germicidal 
for  the  glanders  bacillus.  Guinea-pigs  treated  with  ox- 
serum,  either  before  or  after  infection,  recovered  in  seven 
cases  out  of  ten.  ^lien  inoculated  with  Aery  virulent 
cultures,  Avhich  usually  killed  these  animals  in  five  days, 
the  animals  are  said  to  have  survived  from  twentj'-one 
to  forty-two  days. 

Bonome  (1894)  reports  his  success  in  curing  infected 
guinea-pigs  by  means  of  filtered  cultures  made  in   the 


GLANDERS.  181 

blood-serum  of  the  ox.  He  was  not,  however,  successful 
in  accomplishiug-  this  result  with  mallein  made  in  the 
usual  way. 

BIBLIOGRAPHY. 

Babes,  A.  :  Note  sur  une  substance  isolee  ties  cultures  du  bacille  de  la 

morve.     Arcb.  de  Med.  exper.  et  d'Aiiat.  patholog.,  1892,  No.  4. 
Babes,  A.,  et  Motoc,  A.  M.  :  Sur  les  substances  chimiques  produites  par 

le  bacille  de  la  morve.    Ann.  de  I'lnstitut  de  Patliol.  et  de  Bacteriol. 

de  Bucbarest,  ii.,  63,  1893. 
Bononie,  A.  :  Neue  Beobacbtungen  iiber  die  diagnostiscbe  und  tbera- 

peutiscbe  Wirkung  der  Stollwecbselprodukte  des  Rotzbacillus  bei 

der  Rotzinfektion  des  Menscben  und    der  Tiere.      Dtscbe.  med. 

Wcbscbr.,  1894,  Nos.  86-38,  p.  703,  725,  744. 
Chenot  et  Picq.  :  De  Taction  bactericide  du  serum  de  sang  de  bovides 

sur  le  virus  morveux,  etc.     Compte- rendu,  Soc.  de  Biol.,  1892,  26 

Mars. 
Finger  :  Zur  Frage  der  Immunitat  und  Pbagocytose  beim  Rotz.  Zieg- 

ler's  Beitrage  zur  patbol.  Anat.,  Bd.  VI.,  1889,  Heft  4. 
Fotli  :  Ueber  die  praktiscbe  Bedeutung  des  trockenen  Malleins.     Deut- 

scbe  Zeitscbr.  fur  Tiermed.,  XX.,  223,  1894. 
Die  "  Versucbe  mit  der  Anwendtmg  des  Malleins  in  der  russis- 

chen  Armee."     Centralbl.  f.  Bakteriol,  XVI.,  508,  1894. 

Ueber  Mallein.  Zeitscbr.  f.  Veterinarkunde,  IV.,  435,  1892. 


Hutyra,  F.,  u.  Preisz,  H.  :  Ueber  die  diagnostisclien  Wert  des  Malleins. 
Dtscbe.  Ztscbr.  f.  Tiermed.,  1894,  Bd.  XX.,  p.  309. 

Kresling,  K.  :  Sur  la  preparation  et  la  composition  de  la  malleine. 
Arch.  d.  Sci.  Biolog.,  publ.  par  I'lnst.  Imp.  de  Med.  Exper.  a  St. 
Petersbourg,  1892,  p.  711. 

Preusse  :  Versucbe  mit  Rotzl^nnpbe — Mallein.  Berl.  tbierartzl.  Wcb- 
scbr., 1891,  No.  29. 

Suchanka,  F.  J.  :  Impfversuclie  rait  Mallein.  Mtsb.  f.  prakt.  Tierbeilk., 
1894,  p.  481. 

Scliindelka,  H.  :  Einige  Erfabrungen  tiber  die  Anwendung  des  Mal- 
leins als  diagnostiscbes  Mittel.  Oester.  Ztschr.  f.  wissen.  Veterin- 
arkunde, V.^97,  1894. 

De  Sebweinitz  and  Kilborne  :  The  Use  of  Mallein  for  the  Diagnosis  of 
Glanders  in  Horses,  etc.  Journal  of  Comparative  Med.  and  Vet- 
erinary Archives,  1892,  p.  643. 

Schiitz  :  Malleinversuche.  Arch.  f.  -wissensch.  u.  prakt.  Tierbeilk., 
1894,  No.  6,  p.  448. 

Straus  :  Sur  la  vaccination  centre  la  morve.  Compte-rendu,  Acad, 
des  Sci.,  t.  cviii.,  p.  530. 


YII. 

HOG  CHOLERA. 

Reseaeches  made  by  the  bacteriolog-ists  connected 
with  the  Agricultural  Department  (Bureau  of  Animal  In- 
dustry) have  established  the  fact  that  two  distinct  dis- 
eases of  swine,  due  to  different  bacilli  (one  motile  and 
the  other  not)  prevail  in  epidemic  form  in  various  parts 
of  the  United  States.  The  diff'erentiatiou  of  the  bacillus 
of  hog-  cholera  was  made  in  1885,  and  a  report  by  Theo- 
bald Smith,  relating-  to  this  infectious  disease  of  swine, 
has  recently  been  ijublished  (1894).  The  bacillus  pro- 
duces in  swine  a  disease  in  which  the  principal  pathologi- 
cal lesions  are  found  in  the  large  intestine,  the  mucous 
membrane  of  which  is  more  or  less  ulcerated  and  ne- 
crosed ;  in  some  cases  there  is  at  the  same  time  a  pneu- 
monia of  limited  extent. 

The  bacillus  kills  rabbits,  mice,  and  guinea-pigs  when 
injected  subcutaneously  or  introduced  into  the  stomach. 
A  small  dose  is  fatal  to  a  rabbit  in  about  seven  days, 
but  larger  doses  kill  in  a  shorter  time,  and  it  is  more 
promptly  fatal  when  injected  into  the  circulation.  Sev- 
eral varieties  of  this  bacillus  have  been  encountered  l)y 
the  bacteriologists  of  the  Agricultural  Department ;  these 
differ  chiefly  in  pathogenic  virulence,  and  the  curious 
fact  is  stated  that  the  variety  first  studied,  after  artificial 
cultivation  for  six  and  a  half  years,  has  not  lost  its  patho- 
genic power.     It  still  kills  rabbits  when  injected  into  the 


HOG   CHOLERA.  183 

circulation  in  small  amounts  (0.12  c.c).  All  of  tlie  varie- 
ties closely  resemble  tlie  well-known  Bacillus  coli  commu- 
nis, which  has  its  normal  habitat  in  the  intestine  of 
healthy  men  and  animals.  According-  to  Smith  the  ba- 
cillus {B.  typhi  murium)  discovered  by  Loffler  in  1890, 
which  causes  a  fatal  infectious  disease  in  mice,  is  nearly 
allied  to  the  hog-cholera  bacillus,  and  should  be  included 
in  the  same  group  —  which  may  be  denominated  the 
"  colon  group." 

The  experiments  thus  far  made  with  reference  to  pro- 
tective inoculations  against  hog-  cholera  have  not  given 
very  satisfactory  results.  Selander  and  Metchnikoff  have 
reported  success  in  immunizing  rabbits,  but  according  to 
Smith  their  experiments  were  made  with  the  bacillus  of 
swine  plague,  and  not  with  that  of  hog  cholera  as  they 
supposed.  The  following  conclusions  have  been  formu- 
lated by  Smith  as  a  result  of  his  extended  experiments : 

"  1.  It  is  possible  to  produce  immunity  toward  hog- 
cholera  and  swine-plague  bacteria  in  the  very  susceptible 
rabbit  and  the  less  susceptible  guinea-pig.  In  the  rabbit 
the  only  promising  method  of  immunization  toward  hog- 
cholera  is  the  use  of  gradually  augmented  doses  of  atten- 
uated cultures. 

"2.  Immunization  toward  swine -plague  is  produced 
artificially  with  much  greater  ease  than  toward  hog- 
cholera  bacteria. 

"  3.  The  blood-serum  of  animals  protected  against  hog 
cholera  and  swine  plague  is  almost  as  efficacious  in  pro- 
ducing immunity  soon  after  treatment  as  the  bacterial 
products  obtained  from  cultures. 

"  4.  Different  degrees  of  immunity  in  both  hog  cholera 
and  swine  plague  lead  to  different  forms  of  the  inocula- 
tion disease.  The  greater  the  immunity  short  of  com- 
plete protection  the  more  prolonged  and  chronic  the 
disease  induced  subsequently  by  inoculation. 


184  IMMUNITY    AND   SERUM-TIIERAPY. 

"  5.  Pathogenic  bacteria  may  remain  in  the  org-ans  of 
inoculated  animals  some  time  after  apparently  full  recov- 
ery. Their  presence  may  or  may  not  be  associated  with 
lesions  recognizable  by  the  naked  eye. 

"  6.  The  toxicity  of  sterilized  cultures  appears  to  be  di- 
rectly proportional  to  the  number  of  bacteria  in  the  in- 
jected fluid." 

The  experiments  of  Moore,  reported  in  Bulletin  No.  6  of 
the  Bureau  of  Animal  Industry,  show  that  the  bacillus  of 
hog  cholera  does  not  become  attenuated  by  being  passed 
through  rabbits,  and  that  in  the  experiments  of  Metchni- 
koff,  whicli  led  him  to  conclude  that  this  is  the  case,  the 
bacillus  of  swine-plague,  and  not  that  of  hog  cholera,  was 
used. 

De  Schweinitz  has  studied  the  chemical  products  of 
the  hog-cholera  bacillus  (1890)  and  has  obtained  from  the 
cultures  cadaverin,  methylamin,  a  ptomaine  ("  sucholo- 
toxin"),  and  an  albumose  ("  sucholoalbumiu  "). 

Novy  (1890)  has  also  obtained,  by  Brieger's  method,  a 
basic  toxic  substance  ("  susotoxin  ")  which  kills  rats  in 
the  dose  of  0.125  to  0.25  c.c.  He  also  obtained  from  con- 
centrated cultures,  by  precipitation  with  absolute  alcohol, 
a  toxalbumin  which,  when  dried,  killed  rats  in  three  or 
four  hours  in  the  dose  of  0.05  to  0.01  gm. 

Both  of  the  authors  nam^ed  obtained  experimental  evi- 
dence indicating  that  these  toxic  substances  obtained 
from  cultures,  when  given  to  susceptible  animals  in  non- 
lethal  doses,  cause  them  to  be  immune  from  the  patho- 
genic action  of  small  quantities  of  a  culture  of  virulent 
hog-cholera  bacilli. 

BIBLIOGRAPHY. 

Novy  :  The  Toxic  Products  of  the  Bacillus  of  Hog  Cholera.     Philadel- 
phia Medical  News,  1890,  p.  231. 


HOG   CHOLERA.  185 

Salmon  :  Results  of  Experiments  with  Inoculation  for  the  Prevention  of 
Hog  Cholera.  Farmers'  Bulletin,  No.  8,  Agricultural  Department, 
Washington,  1892. 

De  Schweinitz  :  A  Preliminary  Study  of  the  Ptomaines  from  the  Cult- 
ure Liquids  of  the  Hog-cholera  Germ.  Philadelphia  Medical 
News,  1890,  p.  237. 

Smith  :  Zur  Kenntniss  des  Hogcholerabacillus.  Centralbl.  fiir  Bakte- 
riol.,  Bd.  IX.,  1891,  pp.  253,  307,  339. 

The  Hog-cholera  Group  of  Bacteria.  Bulletin  No.  6,  1894,  Bu- 
reau of  Animal  Industry,  Washington. 


HOG  ERYSIPELAS. 

The  disease  known  in  Germany  as  Rothlauf,  and  in 
France  as  rouget,  is  due  to  a  slender  bacillus,  which  is,  ap- 
parently, identical  with  Koch's  bacillus  of  mouse  septicae- 
mia, which  he  first  obtained  by  inoculating-  mice  with 
putrefying  blood  or  flesh  infusion.  The  bacillus  of  hog- 
erysipelas  {Rothlauf)  was  first  accurately  described  by 
Loffler  and  Scliutz  (1885),  althoug-h  Pasteur  had  previously 
ascribed  the  etiology  of  this  disease  to  a  minute  bacillus 
— no  doubt  the  same — and  had  proved  the  practicability 
of  j)rotecting-  swine  from  the  ravages  of  the  disease  by 
inoculations  with  an  attenuated  virus.  This  bacillus  is 
l^athogenic  for  swine,  rabbits,  white-mice,  house-mice, 
pigeons,  and  sparrows.  Guinea-pig-s,  field-mice,  and 
chickens  are  immune.  Swine  become  infected  hy  the 
ingestion  of  food  containing-  the  bacillus,  and  usually  die 
within  two  or  three  days. 

Pasteur's  first  studies  relating  to  the  etiology  of 
"  i^ougef''  were  made,  in  collaboration  with  Chamber- 
land,  Eoux,  and  Thuillier,  in  1882.  Pasteur  found  that 
the  virulence  of  his  cultures  was  increased  by  passing 
them  through  j)ig-eons  and  diminished  by  passing  them 
through  rabbits.  By  a  series  of  inoculations  in  rabbits 
he  obtained  an  attenuated  virus  suitable  for  protective 
inoculations  in  swine.  In  practice  he  recommended 
the  use  of  a  mild  virns   first,   and  after   an   interval  of 


HOG   ERYSIPELAS.  187 

twelve  days  of  a  stronger  virus.  These  inoculations 
liave  been  extensively  practised  in  France,  and  the 
fact  that  immunity  may  be  established  in  this  way  is 
well  demonstrated.  There  has  been  some  doubt,  how- 
ever, as  to  the  practical  value  of  the  method,  as  its  appli- 
cation has  been  attended  with  some  loss,  and  there  ap- 
pears to  be  danger  that  the  disease  may  be  spread  by 
the  alvine  discharges  of  inoculated  animals.  In  a  region 
where  the  annual  losses  from  the  disease  are  consider- 
able, and  where  the  soil  is,  perhaps,  thoroughly  infected 
with  the  bacilli,  i^rotective  inoculations  probably  afford 
the  best  security  against  loss.  But  when  it  is  practica- 
ble to  stamp  out  the  disease  by  quarantine  of  infected 
animals,  disinfection  of  localities  in  which  cases  have 
occurred,  and  strict  attention  to  cleanliness,  this  will 
probably  be  found  the  best  method  of  combating  the 
malady. 

In  a  recent  paper  (1894)  Chamberland  states  that  in  the 
last  seven  years,  during  which  time  protective  inocula- 
tions have  been  practised  in  France  on  a  large  scale,  the 
mortality  from  rouget  has  been  reduced  to  1.45  per  cent., 
whereas  before  these  inoculations  were  practised  the 
mortality  from  this  disease  was  about  twenty  per  cent. 
Losses  amounting  in  some  instances  to  as  much  as  ten 
per  cent,  have  resulted  from  the  inoculations.  These  are 
ascribed  by  Chamberland  to  secondary  infection,  through 
the  inoculation  wound,  with  other  pathogenic  bacteria. 

Jakobi  (1888)  reports  the  results  of  inoculations  made 
in  1887  and  1888  with  "  vaccines  "  obtained  from  Pasteur's 
agent  in  Paris.  His  results  agree  with  those  previously 
reported  by  Lydtin  in  showing  a  smaller  loss,  as  a  result 
of  the  inoculations,  among  young  pigs  than  among  older 
ones— over  sixteen  weeks.     The  loss  among  young  pigs 


188  IMMUNITY    AND   SERUM-TIIP:RAPV. 

was  only  1.3  per  cent.  The  animals  which  survived  sub- 
sequently escaped  infection,  while  others  not  inoculated, 
associated  with  them,  succumbed  to  the  disease. 

Hut3a'a  has  given  the  following  statistics  of  inocula- 
tions made  in  Hungary  during-  the  year  1889,  with  "vac- 
cines "  obtained  from  the  Pasteur  laborator}^  in  Vienna  : 
48,637  pigs  Avere  inoculated  on  117  different  farms.  Of 
these  143  (0.29  per  cent.)  died  between  the  first  and  sec- 
ond inoculation.  After  the  second  inoculation  59  animals 
died  (0.1  per  cent.).  During  the  year  following  the  inoc- 
ulations 1,082  inoculated  pigs  died  of  RolJilovf.  Before 
the  inoculations  the  annual  loss  in  the  same  localities  is 
said  to  have  been  from  ten  to  thirty  per  cent.  Upon  one 
farm  220  pigs  which  had  been  inoculated  Avere  associated 
with  1,500  not  inoculated.  The  loss  among  the  latter  was 
fifty  per  cent.,  among  the  former  2.27  per  cent. 

In  a  later  communication  (1894)  Jakobi  gives  the  fol- 
lowing results  of  inoculations  made  since  by  the  same 
method :  1889,  inoculated  133,  loss,  5  ;  1890,  inoculated  151, 
loss,  2 ;  1891,  inoculated  158,  loss,  0  ;  1893,  inoculated  223, 
loss,  0  ;  1894,  inoculated  145,  loss,  4.  Total  inoculated, 
1,036  ;  total  loss,  14.  These  inoculations  were  made  upon 
19  different  farms,  and  principally  upon  pigs  less  than 
four  months  old.  The  inoculated  pigs  were  isolated  to 
prevent  the  communication  of  the  disease  to  other  uii- 
protected  pigs. 

Inoculations  with  Blood-serum  of  Immune  Animals. 

The  experiments  of  Lorenz,  commenced  in  1891,  seem 
to  establish  the  fact  that  there  is  an  antitoxin  in  the 
blood  of  animals  which  have  an  acquired  immunity 
against  this  disease  which  may  be  used  for  producing 


HOG   ERYSIPELAS.  189 

immunity  in  other  animals,  or  for  the  cure  of  the  disease 
in  animals  already  infected.  In  his  latest  communica- 
tion (1894)  Lorenz  says  : 

"  When  I  read  in  the  journals  of  the  discovery  of  Beh- 
rinsT  and  Kitasato  that  the  blood  of  animals  immunized 
ag-ainst  tetanus,  when  injected  beneath  the  skin  of  other 
animals,  gave  them  an  immunity  ag-ainst  tetanus,  I  had  in 
my  possession  rabbits  which  were  immunized  against 
Rotldauf.  I  took  from  one  of  these  some  blood  from  the 
ear  vein,  injected  it  under  the  skin  of  a  mouse,  inoculated 
this  later  with  a  Rothlavf  culture,  and  made  the  discovery, 
in  this  and  a  series  of  subsequent  experiments,  that  the 
blood  of  an  animal  immune  against  Rotldauf  contains  an 
immunizing  substance.  I  further  ascertained  that  this 
substance  is  found  only  in  the  blood-serum,  and  not  in 
the  solid  portions  of  the  l)ody  organs,  etc.,  and  with  the 
exception  of  the  blood  was  found  only  in  the  secretions  of 
serous  membranes.  I  also  found  that  the  immunizing 
substance  is  only  to  be  found  for  a  certain  time  after  re- 
newed infection  in  the  immune  animals,  and  that  it  grad- 
ually disappears,  without  the  loss  of  iniTiiunity  in  the 
animal,  however.  Finally,  I  discovered  that  the  animals 
into  which  one  injects  blood-serum  from  immune  animals 
do  not  acquire  a  lasting  immunity,  but  are  only  immune 
for  a  relatively  short  time." 

In  experiments  made  in  1893  and  1894,  with  a  view  to 
producing  immunizing  serum  for  protective  inoculations 
on  a  large  scale,  Lorenz  met  with  some  disappointments ; 
but  he  proposes  to  renew  his  attempts  and  hopes  to  avoid 
the  difficulties  which  have  been  brought  to  light  by  ex- 
perience, one  of  which  he  states  as  follows  : 

"  When  an  animal  already  immunized  against  Rothlavf 
receives  an  injection  of  a  considerable  quantity  of  ds  cult- 
ure of  the  bacillus,  in  order  to  cause  the  production  in 


190  IMMUNITY    AXD   SERUM-THERAPY. 

its  blood  of  a  serum  of  liig-h  therapeutic  value,  the  ani- 
mal bears  these  iujections  without  anj"  notable  reaction. 
But  its  blood-serum  contains  during-  the  following  dajs, 
besides  the  immunizing  substance,  also  poisonous  sub- 
stances, and  blood  which  is  taken  too  soon  (twentj'-four 
hours)  after  the  injection  has  a  toxic  action  upon  animals 
which  are  already  infected.  If  this  poisonous  serum  is 
injected  into  a  mouse  which  has  been  infected  two  days 
before  with  Rothlaufh&itiiW,  in  the  dose  of  about  0.05  c.c, 
death  occurs  in  a  few  hours,  even  when  scarcely  any 
evidence  of  sickness  had  been  observed  before  the  injec- 
tion." 

The  fact  that  mice  infected  with  this  bacillus  may  be 
cured  by  injecting  into  them  blood-serum  from  an  im- 
munized rabbit  has  also  been  demonstrated  by  F.  Klem- 
perer  (1892).  In  his  experiments  with  the  bacillus  of 
mouse  septicaemia,  and  with  Friedlander's  bacillus,  he 
found  that  serum  from  an  immune  rabbit  may  be  used  to 
immunize  mice  and  also  to  cure  them  after  infection, 
while  serum  from  a  non-immune  rabbit  has  no  such  ac- 
tion. The  immunity  produced  in  this  way  was  found  to 
be  specific.  That  is,  animals  immunized  against  the 
pathogenic  action  of  one  of  these  bacilli  were  not  pro- 
tected against  infection  by  the  other.  The  "  heilserum  " 
when  added  to  cultures  in  vitro  did  not  prove  to  have  any 
special  bactericidal  action. 


BIBLIOGRAPHY. 

Emmerich  und  Tsuboi  :  Versuch  der  Immiinisieruug  von  Schweinen 
gegen  Rothlauf.    Dtsche.  tierarztl.  Wchschr.,  1893,  p.  127. 

Hafner  :  Die  Schutzimpfung  gegen  den  Rothlauf  der  Schweine.  Bad. 
thierarztl.  Mitth.,  1889,  p.  17. 

Hess  iind  Guillebeau  :  Zur  Schutzimpfung  gegen  Schweineseuche. 
Schweitzer  Archiv  fiir  Thierheilk.,  Bd.  XXVIII.,  1886,  Heft  3. 


HOG   ERYSIPELAS.  191 

Jakobi  :  Beitrag  zur  Scliutzimpfunggegeii  den  Rotblauf  der  Schweiiie. 

Eerl.  thiei-arztl.  Wchsclir.,  1888,  No.  50. 
Beitrag   zur  Pa.steursclien  Scliutzimpfuiig   gegeu  Rothlauf   der 

Schweine.     Berl.  thieriirztl.  Wcbschr.,  1894,  p.  234. 
Kitl  :  Beitrage  zur  Kenntniss  des  Slabschenrothlauf  der  Scbvveiiie  und 

dessen  Scbutzimpfung.     Revue  ftir  Heilk.  und  Tbierzucbt.,  1886. 
Uiitersucbuiigen  tiber  den  Stabscbenrotblauf  und  dessen  Scbulz- 

inipfung.     Centralbl.  fur  Bakteriol.,  Bd.  II.,  1887,  p.  693. 
Lorcuz  :  Immunisierungversucbe  gegen  Scbweinerolbhuif.    Bad.  tbier- 

arztl,  Mittb.,  1893,  p.  33. 

Ein  Scbutzimpfungsverfabren   gegen    Scbweinerotblauf.     Cen- 

tralltl.  f.  Bakteriol.,  XIII.,  357,  1893. 

Sebutzimpfungsversucbe  gegen   Scbweinerotblauf  mit  Anwen- 

dung  eines  aus  Blutserum  immunisierter  Tiere  hergestellten  Impf- 
praparates.     Dtscbe.  lierarztl.  Wcbschr.,  1894,  p.  9. 

Sebutzimpfungsversucbe  gegen    Scbweinerotblauf.     Centralbl. 


fiir  Bakteriol.,  XV.,  278,  1894. 
Lj'dtin  :  Scbutzimpfungen   gegen   den   Rotblauf   der   Scbweine.    Bad. 

tbierarztl.  :Mitt.,  1886,  No.  9. 
Pasteur  :  Le  rouget  du  pore  ;  avec  la  collaboration  du  MM.  Cbamber- 

land,   Roux  et  Tliuillier.     Conipte-reudu,    Acad,  des   Sci.,   xcv., 

1882,  p.  1120. 
Pasteur  et  Tbuillier  :  Bull,  de  I'Acad.  de  Med.     Paris,  xcvii.,  1883. 
Schiitz  :  Ueber  den  Rotblauf  der  Scbweine  und  die  Impfung  derselbeu. 

Arbeit,  aus  dem  K.  Gesundbeitsamte,  Bd.  I.,  1885. 


IX. 

HYDROPHOBIA. 

Notwithstanding  the  extended  researches  made,  espe- 
cially in  Pasteur's  laboratory,  the  etiology  of  hydro- 
phobia still  remains  unsettled.  It  has  been  demon- 
strated by  experiment  that  the  virus  of  the  disease  is  lo- 
cated in  the  brain,  spinal  marrow,  and  nerves  of  animals 
which  have  succumbed  to  the  disease,  as  w^ell  as  in  the 
salivar}^  secretions  of  rabid  animals ;  and  that  the  disease 
may  be  transmitted  by  intravenous  inoculation,  or  by  in- 
troducing- a  small  quantitj^  of  virus  beneath  the  dura 
mater,  with  greater  certainty  than  by  subcutaneous  in- 
oculations. But  the  exact  nature  of  this  virus  has  not 
been  determined.  The  fact  that  a  considerable  interval 
elapses  after  inoculation  before  the  first  symptoms  are 
developed  indicates  that  there  is  a  multiplication  of  the 
virus  in  the  body  of  the  infected  animal ;  and  this  is  fur- 
ther shown  by  the  fact  that  after  death  the  entire  brain 
and  spinal  marrow  of  the  animal  have  a  virulence  equal 
to  that  of  the  material  with  which  it  was  inoculated  in 
the  first  instance.  The  writer's  experiments  (1887)  show 
that  this  vi^'ulence  is  neutralized  by  a  temperature  of  60° 
C,  maintained  for  ten  minutes — a  temperature  which  is 
fatal  to  all  known  pathogenic  bacteria  in  the  absence  of 
spores.  But  recent  experiments  show  that  certain  toxic 
products  of  bacterial  growth  are  destroyed  by  the  same 
temperature.     We  are,  therefore,  not  justified  in  assum- 


HYDROPHOBIA.  193 

ing-  tliat  the  morbid  phenomena  are  directly  due  to  the 
j)resence  of  a  living-  microorganism  ;  and,  indeed,  it 
seems  probable,  from  what  we  already  know,  that  the 
symptoms  developed  and  the  death  of  the  animal  are  due 
to  the  action  of  a  potent  chemical  poison  of  the  class 
known  as  toxalbumins.  But,  if  this  is  true,  we  have  still 
to  account  for  the  production  of  the  toxic  albuminoid 
substance,  and,  in  the  present  state  of  knowledge,  have 
no  other  way  to  explain  its  increase  in  the  body  of  the 
infected  animal  than  the  supposition  that  a  specific,  liv- 
ing' germ  is  present  in  the  virulent  material,  the  intro- 
duction of  which  into  the  body  of  a  susceptible  animal 
gives  rise  to  the  morbid  phenomena  characterizing-  an 
attack  of  rabies. 

Pasteur  and  his  associates  have  thus  far  failed  to  de- 
monstrate the  presence  of  microorganisms  in  the  virulent 
tissues  of  animals  which  have  succumbed  to  an  attack 
of  rabies.  Babes  has  obtained  micrococci  in  cultures 
from  the  brain  and  spinal  cord  of  rabid  animals,  and 
states  in  his  article  on  hydrophobia  in  "  Les  Bacferies  " 
(second  edition,  p.  791)  that  pure  cultures  of  the  sec- 
ond and  third  generation  induced  rabies  in  susceptible 
animals  ;  but  his  own  later  researches  do  not  appear 
to  have  established  the  etiological  relation  of  this  micro- 
coccus. 

Gibier  (1884)  has  reported  the  presence  of  spherical 

refractive  granules,  resembling  micrococci,  in  the  brain 

of  rabid  animals,  which  he  demonstrated  by  rubbing  up 

a  little  of  the  cerebral  substance  with  distilled  water. 

As  these  supposed  micrococci  did  not  stain  with  the  usual 

aniline  colors  and  were  not  cultivated,  it  appears  very 

doubtful  w^hether  the  refractive  granules  seen  were  really 

microorganisms. 
13 


194  IMMUNITY   AND   SERUM-THERAPY. 

Fol  (1885)  claims  to  have  demonstrated  the  presence  of 
minute  cocci,  0.2  /*  in  diameter,  in  sections  of  spinal  cord 
from  rabid  animals,  by  Weigert's  method  of  staining-. 
The  cords  were  hardened  in  a  solution  of  bichromate  of 
potash  and  sulphate  of  copper,  colored  with  a  solution  of 
hsematoxylon,  and  decolorized  in  a  solution  of  ferrocya- 
nide  of  potash  and  borax. 

The  writer  (1887)  has  made  similar  prei^arations, 
carefully  following-  the  method  as  described  by  Fol, 
but  was  not  able  to  demonstrate  the  presence  of  micro- 
organisms in  the  numerous  sections  made.  Nor  have 
the  observations  of  Fol  been  confirmed  by  the  re- 
searches of  other  bacteriologists  who  have  given  their 
attention  to  the  subject  since  the  publication  of  his 
paper. 

Pasteur  first  announced  his  success  in  reproducing 
rabies  in  susceptible  animals  by  inoculations  of  material 
"  from  the  medulla  oblongata,  the  frontal  lobes  of  the 
cerebral  hemispheres,  and  the  cerebrospinal  fluid  "  in  a 
communication  to  the  Academy  of  Sciences  made  on 
May  30,  1881.  At  the  same  time  he  reported  his  success 
in  the  discovery  of  "  a  method  for  considerably  shorten- 
ing the  period  of  incubation  in  rabies,  and  also  of  repro- 
ducing the  disease  with  certainty."  This  was  by  inocula- 
tions, made  after  trephining,  upon  the  surface  of  the  brain 
with  material  obtained  from  the  brain  of  a  rabid  animal. 
Dogs  inoculated  in  this  way  developed  rabies  in  the 
course  of  two  weeks,  and  died  before  the  end  of  the  third 
week — sometimes  of  furious  rabies  and  sometimes  of  the 
paralytic  form  of  the  disease.  In  a  second  communica- 
tion (December  11,  1882)  Pasteur  reports  his  success  in 
communicating  the  disease  by  the  intravenous  injection 
of  virus  from  the  central  nervous  system  ;  also  the  ex- 


HYDROPHOBIA.  195 

perimental  demonstration  of  the  fact  that  all  forms  of 
rabies  may  be  produced  by  the  same  virus  ;  also  that  all 
portions  of  the  spinal  cord  of  rabid  animals  are  virulent, 
as  well  as  all  parts  of  the  brain  ;  also  that  an  animal 
(dog-)  which  had  recovered  from  a  mild  attack  after  in- 
oculation proved  to  be  subsequently  immune,  and  that 
"  this  observation  constitutes  a  first  step  toward  the 
discovery  of  the  prophylaxis  of  rabies."  On  February  25, 
1884,  many  important  facts  are  stated  which  had  been 
developed  during  the  continuous  study  of  the  disease,  and 
among-  others  the  fact  that  by  passing  the  virus  through 
a  series  of  animals  of  the  same  species  a  fixed  degree  of 
virulence  is  established,  for  .each  susceptible  species,  as 
shown  by  a  definite  and  uniform  period  of  incubation. 
By  this  method  a  virus  had  been  obtained  which  pro- 
duced rabies  in  rabbits  in  seven  or  eight  days,  and  an- 
other which  caused  the  development  of  the  disease  in 
guinea-pigs  in  five  or  six  days  after  inoculation.  In  a 
subsequent  communication  (May  19,  1884)  evidence  is 
given  to  show  that  by  successive  inoculations  in  monkeys 
the  period  of  incubation  is  prolonged,  and  that  the  at- 
tenuated virus  obtained  from  a  monkey,  after  several  suc- 
cessive inoculations  in  this  animal,  when  inoculated  into 
the  dog,  no  longer  produces  fatal  rabies  ;  and  that  dogs 
so  treated  are  subsequently  immune. 

In  his  address  before  the  International  Medical  Con- 
gress at  Copenhagen  (August  11,  1884),  after  a  review  of 
the  facts  developed  during  his  experimental  researches 
made  during  the  preceding  four  years,  Pasteur  gives  an 
account  of  the  test  made  by  a  commission,  appointed  by 
the  Minister  of  Public  Instruction,  to  determine  the  effi- 
cacy of  his  method  as  applied  to  the  protection  of  dogs. 
He  says  that  he  gave  to  the  commission  nineteen  dogs 


196  IMMUNITY    AND   SERUM-THERAPY. 

which  had  been  rendered  refractory  against  rabies  by 
preventive  inoculations.  These  nineteen  dogs  and  nine- 
teen control  animals,  obtained  from  the  pound  without 
any  selection,  were  tested  at  the  same  time.  The  test  was 
made  upon  some  of  the  animals  of  both  series  by  inocu- 
lation with  virulent  material  upon  the  surface  of  the 
brain,  and  upon  others  by  allowing  them  to  be  bitten  by 
rabid  dogs,  and  upon  still  others  by  intravenous  inoc- 
ulations. Not  one  of  the  protected  animals  developed 
rabies ;  on  the  other  hand,  three  of  the  control  dogs  out 
of  six  bitten  by  a  mad  dog  developed  the  disease,  five 
out  of  seven  which  received  intravenous  inoculations  died 
of  rabies,  and  five  which  we^-e  trephined  and  inoculated 
on  the  surface  of  the  brain  died  of  the  same  disease.  In 
a  subsequent  report  the  commission,  of  which  M.  Bouley 
was  president,  stated  that  twenty-three  protected  dogs 
which  were  bitten  by  ordinary  mad  dogs  all  remained  in 
perfect  health,  while  sixty-six  per  cent,  of  the  control 
animals,  bitten  in  the  same  way,  developed  rabies  with- 
in two  months. 

In  his  communication  of  October  26,  1885,  Pasteur  re- 
ports his  discovery  of  the  fact  that  the  virulence  of  the 
spinal  cord  of  a  rabbit  is  gradually  attenuated  by  hang- 
ing it  in  a  dry  atmosphere,  and  is  finally  entirely  lost ; 
also  that  he  had  been  able  to  make  a  practical  applica- 
tion of  this  discovery  in  the  protection  of  dogs  by  means 
of  successive  inoculations  beneath  the  skin  of  an  emul- 
sion of  spinal  marrow  attenuated  in  this  way.  The  first 
inoculation  was  to  be  made  with  a  portion  of  spinal  cord 
which  had  been  kept  long  enough  to  deprive  it  of  all 
virulence,  and  this  was  followed  by  daily  inoculations 
with  more  virulent  material  until,  finally,  material  was 
used  from  a  cord  only  a  day  or  two  old. 


HYDROPHOBrA.  197 

With  reference  to  his  first  inoculations  in  man,  Pasteur 
says: 

"  Making  use  of  tliis  method,  I  had  already  made  fifty 
dogs  of  various  races  and  ages  immune  to  rabies,  and  had 
not  met  Avitli  a  single  failure,  when,  on  the  6th  of  July, 
quite  unexpectedly,  three  persons,  residents  of  Alsace, 
presented  themselves  at  my  laboratory." 

These  persons  were  Theodore  Vone,  who  had  been  bit- 
ten on  the  arm  on  July  ■Ith,  Joseph  Meister,  aged  nine, 
bitten  on  the  same  day  by  the  same  rabid  dog,  and  the 
mother  of  Meister,  who  had  not  been  bitten.  The  child 
had'been  thrown  down  by  the  dog  and  bitten  upon  the 
hand,  the  legs,  and  the  thighs,  in  all  in  fourteen  different 
places.  Pasteur  commenced  the  treatment  on  July  6th, 
by  injecting  beneath  the  skin  of  this  child  an  emulsion  of 
cord  which  had  been  kept  for  fourteen  days;  this  was 
followed  by  twelve  more  inoculations  made  on  successive 
days  with  cord  of  increasing  degrees  of  virulence — the 
last  with  cord  a  day  old.  On  March  1,  1886,  Pasteur  re- 
ported to  the  Academy  of  Sciences  the  fact  that  the  boy 
Meister  remained  in  good  health  and  gave  detailed  infor- 
mation with  reference  to  a  number  of  cases  which  had 
since  been  treated  by  the  same  method. 

With  reference  to  the  duration  of  the  immunity  result- 
ing from  these  inoculations  Pasteur  says  (188G)  that  out 
of  fourteen  dogs  inoculated  with  "  ordinary  street  virus, ' 
by  trephining,  at  the  expiration  of  a  year  after  the  pro- 
tective inoculations  had  been  practised,  eleven  resisted ; 
out  of  six  tested  in  the  same  way  at  the  end  of  two  years 
two  proved  to  be  immune. 

In  November,  1886,  Pasteur  communicated  to  the  Acad- 
emy of  Sciences  the  results  of  his  experiments  with  refer- 


198 


IMMUNITY   AND   SERUM-THERAPY. 


ence  to  a  modification  of  his  metliod  as  at  first  employed 
— the  so-called  intensive  method.  This  modification  con- 
sisted in  making  the  inoculations  with  cords  of  increasing 
virulence  in  more  rapid  succession. 

The  method  followed  at  Odessa,  as  reported  by  Gama- 
leia  (1887),  is  shown  below,  the  day  being  given  above  and 
age  of  the  cord  below. 


1 


2 


3 

4 

5 

6 

1 

8 

9 

10 

10-9 

8-7 

6-5 

4-3 

2-10 

8-6 

4 

2 

14-13      12-11 

Since  the  adoi^tion  of  this  method  and  the  use  of 
larger  quantities  of  virus,  according  to  Gamalei'a,  there 
have  been  no  deaths  among  those  inoculated,  numbeadng 
more  than  two  hundred  at  the  time  the  report  was  made. 
The  author  last  referred  to  concludes  from  his  experience 
that  "  the  mortality  diminishes  in  direct  relation  to  the 
quantity  of  the  vaccine  injected." 

Bujwid  (1889)  reports  a  total  of  670  inoculations,  with  9 
deaths,  made  at  Varsovio  during  the  years  1886, 1887,  and 
1888.     His  method  is  shown  below. 


12-10 


8-6' 


The  results  of  inoculations  made  at  the  Pasteur  Insti- 
tute in  Paris  during  the  years  1886  to  1890  are  given  in 
the  following  table  : 


Year; 

Number  Treated. 

Died. 

MortaUty. 

1886 

2,671 
1,770 
1,622 
1,830 
1,540 

25 

13 

9 

6 

5 

0.94 

1887 

0.73 

1888 

0.55 

1889 

0.83 

1890 

0.32 

Total 

9,433 

58 

0.61 

HYDROPHOBIA. 


199 


In  the  following-  table,  A  includes  all  persons  treated 
wlio  had  been  bitten  by  an  animal  proved  to  be  rabid ;  B, 
persons  bitten  by  animals  examined  by  veterinary  snr- 
g-eons  and  pronounced  rabid ;  C,  persons  bitten  by  ani- 
mals suspected  of  being-  rabid.  The  figures  relate  to  the 
year  1890  : 


Number  Treated. 

Died. 

Mortality. 

A 

416 
909 
215 

0 
4 
1 

B 

0.44 

(; 

0  46 

Bordoni-Uffreduzzi  g-ives  the  following  statistics  with 
reference  to  the  inoculations  practised  at  the  Pasteur  In- 
stitute in  Turin  during  the  years  1886  to  1891 :  81  persons 
were  inoculated  by  the  method  first  proposed  by  Pasteur, 
with  a  mortality  of  2.46  per  cent.;  925  persons  were  subse- 
quently inoculated  by  the  same  method,  but  with  larg-er 
doses  of  virus,  with  a  mortality  of  1.72  per  cent.  Finally, 
338  persons  were  inoculated  with  still  larg-er  doses,  with 
a  mortality  of  0.29  per  cent. 

At  the  Pasteur  Institute  in  Palermo  the  number  of  per- 
sons inoculated  in  the  four  years  prior  to  1891  was  662, 
with  a  mortality  among  the  inoculated  of  0.6  per  cent.  In 
Bologna  (1890)  210  persons  bitten  by  dog's  undoubtedly 
mad  were  inoculated,  with  a  mortality  of  0.47  per  cent. 

In  the  Pasteur  Institute  at  Naples  810  persons  were 
treated  during  the  years  1886  to  1892,  with  a  mortality  of 
0.86  per  cent. 

During  the  year  1891,  1,564  persons  were  inoculated  at 
the  Pasteur  Institute  in  Paris,  with  a  total  mortality  of 
0.57  per  cent.  In  324  of  these  cases  the  animal  which  in- 
flicted the  bite  was  proved  to  be  rabid  by  experimental 
inoculations. 


200  IM3IUNITY   AND   SERUM-THERAPY. 

Horsely  (1889)  lias  made  a  comparison  of  the  results 
obtained  by  the  "  intensive  treatment "  as  compared  with 
those  by  the  treatment  first  employed,  and  says  : 

"It  is  evident  that  the  intensive  treatment  is  very  suc- 
cessful in  coping  with  the  worst  cases,  and  that,  instead 
of  being-  itself  a  source  of  death,  as  asserted  by  those  who 
gain  notoriety  and  subsistence  by  vilifying  and  misrep- 
resenting scientific  progress,  it  is  a  powerful  agent  in 
saving  life." 

The  following  table  is  given  by  Horsely  "  as  showing 
the  contrast  between  the  old  or  simple  treatment  and 
the  intensive  treatment :  " 

Simple  Treatment,  1886.  Intensive  Treatment,  1888. 

Odessa 3.39  per  cent.  0.64  per  cent. 

Warsaw 4.1       "      "  0.0*    "      " 

Moscow 8.2f    "      "  1.6      "      " 

Perdrix  (1890),  in  an  analysis  of  the  results  obtained  at 
the  Pasteur  Institute  in  Paris,  calls  attention  to  the  fact 
that  the  mortality  among-  those  treated  has  diminished 
each  year  and  ascribes  this  to  improvement  in  the  meth- 
od.   He  says  : 

"  At  the  outset  it  was  difiicult  to  know  what  formula  to 
adopt  for  the  treatment  of  each  particular  case.  Uj)on 
consulting  the  accounts  of  the  bites  in  persons  who  have 
died  of  hydrophobia  notwithstanding  the  inoculations, 
we  have  arrived  at  a  more  precise  determination  as  to  the 
treatment  suitable  for  each  case,  according  to  the  gravity 
of  the  lesions.  In  the  cases  with  serious  wounds  we  in- 
ject larger  quantities  of  the  emulsion  of  cord  and  repeat 

*  The  figures  include  sixteen  months'  work,  and  thirtj-  individuals  bitten  in 
the  face — four  by  wolves. 

+  This  unusually  high  rate  was  found  to  be  due  to  imperfections  in  the  man- 
ner of  preparing  the  cords  for  the  inoculation  material. 


HYDROPHOBIA.  201 

the  inoculations  with  the  most  virulent  material.  For  the 
bites  upon  the  head,  which  are  especially  dangerous, 
however  slight  their  apparent  gravity  may  be,  the  treat- 
ment is  more  rapid,  and,  above  all,  more  intensive — that 
is  to  say,  the  virulent  cord  is  injected  several  times." 

The  statistics  arranged  with  reference  to  the  location 
of  the  bite  are  given  by  Perdrix  as  follows  : 

Bitten  upon  the  head,       684  ;  died,  12,  =  1.75  per  cent. 
"  hands,  4,396;     "        9,  =  0.2      "      " 
"  limbs,   2,839;     "        5,  =  0.17    "      " 

Other  methods  of  making  susceptible  animals  immune 
against  hydrophobia  have  been  proposed  and  proved  by 
experiment  to  be  successful.  Thus  Galtier  in  1880-1881 
claimed  that  the  sheep  and  the  goat  could  be  protected 
by  intravenous  injections  of  the  virus  of  rabies,  and  more 
recent  experiments  fully  confirm  this.  Protopopoff  (1888) 
by  injecting  an  emulsion  of  cord  from  a  rabid  animal  into 
the  circulation  of  dogs  succeeded  in  protecting  them  from 
hydrophobia  as  a  result  of  a  subsequent  inoculation  with 
virulent  material  upon  the  surface  of  the  brain.  He  in- 
jected into  a  vein,  at  intervals  of  three  daj's,  1  c.c.  of  an 
emulsion  of  cord — first  of  six  days,  second  of  three  days, 
third  of  one  da3^  Roux  had  previously  accomplished 
the  same  result  by  a  single  intravenous  injection  of  a 
larger  quantity  (35  c.c.)  of  cord  which  had  been  kept  for 
five  or  six  days.  In  discussing  his  results  Roux  calls 
attention  to  the  fact,  which  had  been  developed  during 
his  experiments,  that  the  virulence  of  the  spinal  cord  of 
rabid  animals  does  not  depend  entirely  upon  the  length  of 
time  it  has  been  kept,  but  that  large  doses  of  cord  kept  as 
long  as  twelve  days  will  sometimes  produce  hydrophobia 
when  injected  into  the  circulation  of  dogs,  when  smaller 
doses  of  cord  kept  five  or  six  days  prove  to  be  inofi'eusive. 


202  IMMUNITY   AND   SERUM-THERAPY. 

He  supposes  that  during-  desiccation  the  virus  may  not 
be  equally  acted  upon  throughout  the  cord,  but  that  cer- 
tain "  islands  "  in  the  centr.  J  portion  may  remain  living- 
and  ^druleut  when  all  the  rest  has  been  modified.  A 
IDractical  point  with  reference  to  the  preservation  of  vir- 
ulent material  is  referred  to  by  Roux  in  a  note  published 
in  the  "  Annales  of  the  Pasteur  Institute."  This  is  the 
fact  that  when  preserved  in  glycerine  jDortions  of  the  cen- 
tral nervous  system  retain  their  virulence  for  considera- 
ble time.  Other  forms  of  virus,  e.g.,  vaccine,  may  also  be 
preserved  in  the  same  way. 

Centanni  (1892)  has  succeeded  in  making  rabbits  im- 
mune by  inoculating  them  with  an  attenuated  virus  ob- 
tained by  subjecting  virulent  material  to  the  action  of  an 
artificial  gastric  juice.  After  digestion  for  less  than 
twelve  hours  the  virus  still  kills  rabbits,  when  inoculated 
beneath  the  dura  mater,  but  the  period  of  incubation  is 
considerably  prolonged.  After  from  twelve  to  twenty 
hours'  digestion  it  no  longer  kills  ral)bits,  but  causes  an 
infection,  from  which  they  recover,  and  after  which  they 
are  immune. 

Sent  n  t-the  >  ajyy. 

Tizzoni  and  Centanni  (1892)  have  reported  success  in 
the  treatment  of  infected  rabbits  by  the  use  of  blood- 
serum  from  immune  animals  of  the  same  sjiecies — immu- 
nized by  the  "  Italian  method  "  above  described.  The 
animals  experimented  upon  were  inoculated  with  a 
"  street  virus  "  which  produced  paralytic  rabies  in  rab- 
bits and  caused  their  death  in  from  fourteen  to  eighteen 
days.  The  blood-serum  was  obtained  from  rabbits  which 
had  been  proved  to  be  immune  by  resisting  inoculations 


HYDKOPHOBIA.  203 

of  virus  of  full  strength  on  tlie  surface  of  the  brain.  The 
blood-serum,  in  doses  of  3  to  5  c.c,  was  injected  subcu- 
taneously,  or  into  the  peritoneal  cavity,  or  into  the  circu- 
lation. Injections  were  made  into  each  animal  (in  all 
from  11  to  26  c.c.)  after  the  first  symptoms  of  paralytic 
rabies  had  appeared  (on  the  7th,  10th,  lltli,  and  14th  day 
after  infection).  Four  rabbits  treated  in  this  way  fully 
recovered.  In  a  subsequent  experiment  the  bacteriolo- 
g-ists  named  treated  three  rabbits  with  a  dry  antitoxin 
obtained  by  precipitation  from  the  blood-serum  of  im- 
mime  rabbits.  The  precipitate  was  obtained  by  adding- 
1  part  of  serum  to  10  parts  of  alcohol,  and  was  dried  171 
vacuo.  This  dried  precipitate,  in  doses  of  0.18  to  0.25  gm., 
was  dissolved  in  sterilized  water  and  injected  as  in  the 
previous  experiment.  Commencing-  on  the  eighth  day 
after  infection  five  or  six  doses  were  g-iven — in  all  0.9  to 
1.3  g-m.  All  of  the  animals  treated  recovered,  wdiile  all  of 
the  control  animals  died.  Babes  had  previously  (1889) 
reported  successful  results  in  conferring-  immunity  upon 
susceptible  animals  by  injections  of  blood-serum  from 
immune  animals. 

Tizzoni  and  Schwartz,  in  pursuing  this  line  of  investi- 
gation (1892),  report  that  while  the  blood-serum  of  im- 
mune rabbits  neutralizes  the  "  fixed  virus  "  of  rabies  m 
vifi'o,  after  short  contact  (five  hours),  the  blood-serum  of 
immune  dogs  has  but  slight  antitoxic  potency.  The  im- 
munizing substance  in  the  rabbit  serum  does  not  dialyze, 
is  soluble  in  g-lycerin,  is  precipitated  by  alcohol,  and  in 
general  behaves  like  a  globulin.  In  subsequent  experi- 
ments Tizzoni  and  Schwai-tz  used  blood-serum  from  dogs 
and  rabbits  immunized  by  Pasteur's  method.  The  blood 
was  drawn  from  the  carotid  of  the  immune  animals,  and 
the  serum  from  the  same,  mixed  with  virulent  spinal  mar- 


204  IMMTTNITY    AT^D   SERUM-TIIEKAPY. 

row  in  the  form  of  a  liomog-eneous  emulsion,  obtained  by 
crushing'  and  pressing-  through  linen.  These  experiments 
corresponded  with  those  iireviously  made  as  to  the  supe- 
rior antitoxic  power  of  rabbit-serum,  which,  after  five 
hours'  contact,  neutralized  the  virulence  of  the  emulsion  of 
cord.  By  the  injection  of  serum  from  an  immune  rabbit, 
in  doses  of  5  c.c,  into  the  circulation  of  other  rabbits,  they 
were,  as  a  rule,  made  immune.  The  immunizing-  sub- 
stance (antitoxin)  was  shown  by  other  experiments  to 
be  present  only  in  the  blood.  Extracts  from  the  liver, 
spleen,  kidneys,  or  muscles  gave  a  negative  result. 

In  their  latest  communication  (1894)  Tizzoni  and  Cen- 
tanni  give  an  account  of  further  exj)eriments  made  prin- 
cipally upon  sheep  and  dogs.  By  repeated  inoculations 
they  succeeded  in  obtaining  from  these  animals  a  serum 
having  an  immunizing  value  of  1  to  25,000  or  more,  and 
from  this  a  precipitate  was  obtained  estimated  to  have  a 
value  of  1  to  300,000,  and  which  in  doses  of  0.23  gm.  (of 
the  dried  precipitate),  dissolved  in  five  times  its  weight 
of  water,  ought  to  be  a  sufficient  dose  to  protect  a  man 
from  the  development  of  hydrophobia  after  being  bitten 
by  a  rabid  animal. 

The  authors  named  believe  that  inoculations  with  this 
antitoxin  would  be  reliable  for  man,  and  that  they  would 
possess  decided  advantages  over  Pasteur's  method  of  in- 
oculation.    These  advantages  are  specified  as  follows : 

"  Applicability  at  any  time  during  the  period  of  incu- 
bation up  to  the  moment  of  the  appearance  of  symptoms 
of  rabies  ;  absolute  absence  of  virulence  and  of  any  in- 
jurious action  ;  very  rapid  treatment  by  the  injection  of 
one  or  several  small  doses  of  material  ;  complete  solubil- 
ity and  consequently  jirompt  absorption  of  the  material 
injected  and  its  easy  preservation  in  a  dry  condition." 


HYDROPHOBIA.  205 

Finally,  the  authors  say  that  they  are  engaged  in  pre- 
paring the  antitoxin  on  a  sufficient  scale  to  enable  the 
test  to  be  made  upon  man,  and  that  for  this  purpose  they 
are  using  sheep  as  the  most  suitable  animals  for  the  pur- 
pose. 

BIBLIOGRAPHY. 

Babes  et  Lepp  :  Reclierches  siir  la  vaccination  antirabique.  Ann.  de 
I'lust.  Pasteur,  vol.  iii.,  1889,  p.  384. 

Babes,  V.  :  Ueber  die  ersten  erfolgreiclien  Iinpfungen  gegen  Huuds- 
wutli  mittels  Blut  immunisirter  Thiere.  Dtsche.  med.  Wchsclir., 
1893,  No.  41. 

Bardacli :  Sur  la  vaccination  intensive  des  chiens  inocules  de  la  rage 
par  trepanation.     Ann.  de  I'lnst.  Pasteur,  vol.  i.,  1887,  p.  84. 

Bujwid  :  La  methode  Pasteur  a  Varsovie.  Ann.  de  I'lnst.  Pasteur, 
1889,  p.  177. 

Centanni,  E.  :  II  Metodo  italiano  di  vaccinazione  antirabbica.  Ri- 
forma  Med.,  1893,  Nos.  103,  103,  104. 

Die  spezifiscbe  Immunisation  der  Elemente  der  Gevvebe.     Ein 

Beitrag  zur  Keuntniss  der  Immunitat  und  der  Serumtherapie  bei 
Rabies.     Dtscbe.  med.  Wchscbr.,  1893,  pp.  1061,  1115. 

Gamaleia  :  Vaccination  antirabique  des  animaux.  Ann.  de  I'lnst.  Pas- 
teur, vol.  i.,  1887,  pp.  137,  296. 

Galtier  :  Nouvelles  experiences  tendant  a  demontrer  I'efflcacite  des  in- 
jections intra-veineuses  de  virus  rabique.  Compte-rendu,  Acad, 
des  Sci.,  evil.,  1888,  p.  798. 

Nouvelles  experiences  sur  I'inoculation  antirabique,  etc.  Compte- 
rendu,  Acad,  des  Sci.,  1888,  and  cvi.,  p.  1189. 

Hogyes,  A.  :  Vaccinations  centre  la  rage  avant  et  apres  infection.  Ann. 
de  I'lnst.  Pasteur,  t.  iii.,  1889,  p.  439. 

Die  Statistik  des  dritten  Jahres  am  Budapester  Pasteur-Institut. 

Ungar  Arcb.  f.  Med.,  1894,  p.  1. 

Horsley,  Victor  :  On  Rabies,  its  Tieatment  by  M.  Pasteur,  and  on  tbc 
Means  of  Detecting  it  in  Suspected  Cases.  British  Medical  Jour, 
nal,  Feb.  16,  1889,  p.  343. 

Nocard  et  Roux  :  Experiences  sur  la  vaccination  des  ruminants  coU' 
tre  la  rage,  par  injections  intra-veineuses  de  virus  rabique.  Ann. 
de  I'lnst.  Pasteur,  vol.  ii.,  1888,  p.  341. 

Pasteur  :  Sur  la  methode  de  prophylaxie  de  la  rage  apres  morsure. 
Compte-rendu,  Acad,  des  Sci.,  t.  cviii.,  1889,  p.  1338. 

Pasteur,  Chamberland  et  Roux  :  Methode  pour  prevenir  la  rage  aprfis 
morsure.     Compte-rendu,  Acad,  des  Sci.,  1885  (October  36th). 


206  IMMUNITY    AND   SERUM-THERAPY. 

Peter  :  Les  vaccinations  antirabiques.     Journal  de  Micrographie,  1887, 

p.  449. 
Perdrix  :  Les  vaccinations  antirabiques  a  I'Institut  Pasteur.     Ann.  de 

rinst.  Pasteur,  vol.  iv.,  1890,  p.  129. 
Protopopoff  :  Zur  Immunitat  fur  Tolhvutligift  bei  Huuden.     Centralbl. 

fiir  Bakteriol.,  Bd.  IV.,  1»88,  p.  85. 
Ueber  die  vaccination  der  Hundc  gegeu  Tollwutb.     Centralbl. 

fur  Bakteriol.,  Bd.  IV.,  1888,  p.  787. 
Ueber  die  Hauplursacbe  der  Abschwacbung  des  ToUwutbgiftes. 


Centralbl.  fiir  Bakteriol.,  Bd.  VI.,  1889,  p.  129. 
Roux  :  Note  sur  un  moyen  de  couserver  les  moelles  rabiques  avec  leur 

virulence.     Ann.  de  I'lnst.  Pasteur,  vol.  i.,  1887,  p.  87. 
Note  de  laboratoire  sur  I'iinmunite  conferee  aux  cbiens  contre 

la  rage  par  I'iujectiou  intra-veineuse.     Ann.  de  I'Inst.  Pasteur,  vol. 

ii.,  p.  479. 
Suzor  :  Hydropbobia  ;  An  Account  of  M.  Pasteur's  System,  containing 

a  Translation  of  all  bis  Comm.uuicatious  on  tbe  Subject,  tbe  Tecb- 

niqiie  of  bis  Metbod,  and  tbe  latest  Statistical  Results.     London, 

1887,  pp.  231. 
Tizzoni,  G.,  e  Centanui,  E.  :  Ulteriori  ricbercbe  suUa  cura  della  rab- 

bia  sviluppata.     Riforma  Med.,  1892,  No.  182. 
Siero  antirabbico  ad  alto  potere  immunizzante,  applicabile  all' 

uomo.     Riforma  Med.,  1893,  p.  855. 

Serum  gegeu   Rabies,  von  bober    immunisierender    Kraft,  auf 


den  Menscben  anwendbar.     Berl.  klin.  Wocbenscbr.,  1894,   p.  189. 
Tizzoni  et  Scbwartz :  La  prophylaxie  et  la  guerison  de  la  rage  par  le 

sang  des  animaux  vaccines  contre  cette  maladie.     Ann.  de  Micro- 

grapbie,  t.  iv.,  1892,  p.  169. 
La  Profilassi  e  la  cura  dolla  rabbia  col  sangue  degli  animali  vac- 

cinati  contro  quella  malattia.     Riforma  Med.,  1892,  Nos.  18  and  19. 
Ullmann  :  Ein  Beitrag  zur  Frage  tiber  den  Wertb  der  Pasteur'scben 

Scbutzinipfungen  am  Menscben.     Wiener  Med.   Blatter,  1887,  p. 

1260. 
Vulpian  :  Nouvelle  statistique  des  per.sonnes  qui  ont  ete  traitees  a.  I'in- 

stitut  Pasteur,  etc.     Compte-rendu,  Acad,  des  Sci.,  1887  (January 

28tb). 
Wilke  :  Resultate  der  Pasteur'scben  Scbutzimpfung  in  russischen  Insii- 

tuten.     Hygien.  Rundschau,  1894,  No.  20,  p.  920. 


X. 

INFLUENZA. 

The  bacillus  discovered  by  Pfeiffer,  in  1892,  is  now 
well  established  as  the  specific  cause  of  this  disease. 
Bruschettini  has  recently  (1893)  reported  the  details  of 
his  experiments  upon  rabbits,  for  which  animals  this  bacil- 
lus is  pathog-enic.  As  a  result  of  these  experiments  he 
has  reached  the  following-  conclusions  : 

"  1.  Eabbits  may  be  vaccinated  ag-ainst  the  pathogenic 
action  of  cultures  of  the  influenza  bacillus  without  g-reat 
difliculty. 

"  2.  The  best  material  for  producing  a  liig-h  grade  oi" 
immunity  is  blood-cultures  which  have  been  filtered 
through  the  Berkenfeld  filter. 

"  3.  The  blood-serum  of  immunized  animals  has  strong 
antitoxic  properties,  but  has  no  germicidal  power. 

"  4.  The  serum  of  vaccinated  animals  has  the  power  of 
conferring  immunity  upon  other  animals,  in  compara- 
tively small  amounts — in  the  proportion  of  1  to  42,000  of 
body-weight,  and  perhaps  still  less. 

"  5.  This  serum  has  also  a  decided"'  curative  action,  and 
rescues  rabbits  from  death  even  as  late  as  forty-eight 
hours  after  infection  by  injection  of  a  culture  of  the  bacil- 
lus into  the  trachea." 

These  results  lead  the  author  to  hope  that  serum-ther- 
apy'may  afford  a  method  of  curing  this  disease  in  man. 
For  this  purpose  the  blood  of  an  immune  rabbit  would 


208  IMMUNITY   AND   SERUM-THERAPY. 

appear  to  be  the  most  promising  source  from  which  to 
procure  an  antitoxic  serum. 

BIBLIOGRAPHY. 

Bruscliettini :  Nuovo  contribulo  alio  studio  del  bacillo  dell'  iuflueuza 
e  specialmente  della  sua  azione  patogena  vel  couiglio.  La  Riformu 
Med.,  1893,  p.  8L 


XI. 

INFLUENZA  IN  HORSES. 

ScHUTZ  (1887)  has  described  a  minute  oval  bacillus, 
usually  associated  in  pairs,  which  appears  to  be  the  spe- 
cific infectious  agent  in  the  disease  known  in  Germany  as 
Brustseuclie.  This  bacillus  is  pathogenic  for  mice,  rab- 
bits, pigeons,  and  guinea-pigs,  but  not  for  swine  or 
chickens.  By  injection  of  cultures  into  the  parenchyma 
of  the  lungs  Schiitz  reproduced  the  disease — confirmed  in 
1888  by  Hell. 

Horses  which  have  suffered  an  attack  of  infectious  in- 
tluenza  are  subsequently  immune,  and  the  experiments  of 
Hell  have  shown  that  an  immunity  also  follows  the  dis- 
ease which  results  from  inoculations  with  pure  cultures 
of  the  Schiitz  bacillus. 

The  extended  experiments  made  by  the  War  Depart- 
ment of  the  German  Government  show  that  the  disease 
is  not  produced  by  intravenous  injections  or  by  the  in- 
gestion of  the  bacillus  with  the  food.  Infection  occurs, 
however,  when  cultures  are  injected  into  the  respiratory 
passages.  Subcutaneous  injections  cause  a  painful  local 
tumefaction,  often  followed  by  an  abscess,  but  without 
the  general  symptoms  of  influenza. 

Experiments  have  been  made  in  Germany,  by  Hell, 
Siedamgrotzki,  and  others,  which  indicate  that  the  sub- 
cutaneous injection  of  blood-serum  from  immune  horses 

may  confer  immunity  on  other  horses.     Hell  usually  in- 
14 


210  IMMUNITY    AND   SERUM-TIIERAPY. 

jected  40  c.c.  at  a  time,  and  repeated  this  at  intervals  un- 
til 200  to  240  c.c.  had  been  injected  in  the  course  of  two 
or  three  weeks.  He  also  reports  the  results  of  treatment 
by  injections  of  blood-serum  into  the  trachea  in  horses 
already  infected,  and  thinks  these  injections  had  a  favor- 
able influence  on  the  course  of  the  disease.  Experiments 
made  subsequently  by  Toepper  have  given  a  similar  re- 
sult, but  others  have  not  been  so  fortunate,  and  the  im- 
munizing- value  of  blood-serum  injections,  as  practised  by 
the  authors  referred  to,  seems  to  be  still  a  matter  of  some 
doubt.  Toepijer  (1893)  gives  full  directions  for  collecting 
the  serum  and  a  detailed  account  of  results  of  experimen- 
tal inoculations  made  by  himself  and  others.  He  prefers 
to  inject  the  serum  into  the  breast  over  the  ensiform  car- 
tilage.    No  reaction  occurs  after  the  injection. 

BIBLIOGRAPHY: 

Bnischettini,    A.  :     Die   experimentelle   Immuuitat    gegen    Influenza. 

Dtscbe.  med.  Wcbschr.,  1893,  p.  790. 
Hell  :    Immunisirungsversuche    mit    Blutserum    gegen    Brustseiiche. 

Zeitschr.  f.  Veterinark.,  IV.,  453,  527,  1892. 
Schiitz  :  Weitere  Mittheilungen  iiber  Impfungen  mit  Blutserum  zum 

Scliutze  gegen  die  Brustseuche.     Zeitschr.  ffir  Veterinarkunde,  Bd. 

v..  Heft  5. 
Toepper,  P.:  Die  Brustseuche  der  Pferde  und  die  Bekampf ung  dersel- 

ben  durcb  Impfung.     Berl.  tierarztl.  Wcbschr.,  1893,  pp.  342,  853. 


xn. 

PLEURO-PNEUMONIA   OF   CATTLE. 

In  a  recent  communication  (1894)  to  the  Central  Society 
of  Veterinary  Medicine  (of  France),  Arloing-  claims  that 
he  has  demonstrated  the  etiological  relation  of  a  bacillus 
lirst  described  by  him  in  1889  {Pnemjiohadllus  liquefaciens 
hovis)  to  the  infectious  disease  of  cattle  known  as  pleuro- 
pneumonia. The  demonstration  was  not  complete  until 
recently,  because  of  failure  to  reproduce  the  disease  by 
inoculation  with  a  pure  culture  of  the  bacillus.  Arloing- 
asserts  that  he  has  now  succeeded  in  accomx)lishing-  this, 
and  ascribes  his  earlier  failures  to  the  fact  that  the  bacilli 
in  his  cultures  were  too  much  attenuated  to  produce  the 
disease  by  inoculation.  He  has  found  that  the  bacilli  as 
found  in  the  serum  from  the  lung-s  become  more  virulent 
when  they  develop  in  the  subcutaneous  tissues  of  an  ani- 
mal ;  and  cultures  made  from  the  bacillus  procured  from 
the  subcutaneous  tissues  at  the  point  where  protective  in- 
oculations are  usually  practised — the  tail — proved  to  be 
sufficiently  active  to  reproduce  the  disease  when  they 
were  injected  into  the  lung-s.  Chauveau,  who  was  pres- 
ent at  the  meeting-  when  Arloing  read  his  most  recent 
paper  on  the  subject  (June  28,  1894),  states  that  he  has 
seen  the  inoculated  animals  and  has  no  doubt  that  the 
bacillus  in  question  is  the  infectious  agent. 

Although  this  demonstration  is  of  such  recent  date, 
protective  inoculations  against  this  disease   have  long 


212  IMMUNITY    AND   SERUM-THERAPY. 

been  successfully  practised.  For  this  purpose  serum 
obtained  from  the  lungs  of  an  animal  recently  dead  has 
been  employed,  this  having-  been  proved  by  exi^eriment 
to  be  infectious  material  altlioug-h  the  exact  nature  of  the 
infectious  agent  present  in  it  was  not  determined. 

Willems,  who  was  one  of  the  first  to  advocate  the  use 
of  protective  inoculations  in  this  disease  (1852),  has  re- 
cently given  a  lecture  (1894)  in  which  he  has  reviewed  the 
evidence  in  favor  of  these  inociilations  in  the  disease  under 
consideration.  Various  methods  have  been  employed. 
Thus  Willems  states  that  the  natives  of  the  banks  of  the 
Zambeze  cause  animals  to  swallow  a  certain  quantity  of 
the  liquid  from  the  pleural  cavity  of  an  animal  recently 
dead,  and  thus  g-ive  them  immunity.  The  virus  has  been 
injected  into  the  circulation  by  some  experimenters,  and 
others  have  proposed  to  attenuate  it  by  heat.  But  the 
method  which  has  been  most  extensively  employed  is 
that  discovered  by  the  Dutch  settlers  at  the  Cape  of 
Good  Hope  (the  Boers),  and  consists  in  inoculating-  ani- 
mals in  the  tail  with  serum  from  the  lung-s  of  an  animal 
recently  dead  ;  or  with  a  virus  obtained  from  the  tume- 
faction produced  by  such  an  inoculation  in  the  tail.  This 
secondary  virus  was  very  extensively  used  by  Lenglen,  a 
veterinarian  at  Arras,  who  communicated  his  results  to 
the  Academy  of  Science  at  Paris,  in  April,  1863,  and  Wil- 
lems says,  in  his  last  published  communication,  that  this 
is  the  method  which  he  prefers.  It  is  also  the  meth- 
od most  extensively  employed  in  Australia,  into  which 
country  infectious  pleuro-pneumonia  was  introduced  in 
1858.  It  quickly  spread  and  has  caused  enormous  losses. 
The  killing  of  all  animals,  sick  or  suspected  of  being 
infected,  was  tried  for  several  years  ;  but  this  proved  to  be 
ineffectual  for  stamjiing-  out  the  disease,  and  the  sacrifice 


PLEURO-PNEUMONIA    OF    CATTLE.  213 

was  SO  great  that  this  measure  of  prophylaxis  was  aban- 
doned. 

According-  to  Loir,  attention,  in  Australia,  was  called  to 
Willem's  method  of  protective  inoculations,  in  1861,  by  a 
letter  from  Cape  Colony  published  in  the  journals  of  Syd- 
ney and  in  Melbourne.  "  The  method  was  at  once  applied 
both  in  Victoria  and  in  New  South  Wales,  and  since  that 
date  many  thousands  of  cattle  have  been  inoculated.  In 
order  to  obtain  a  sufficient  supply  of  virus  the  method 
recommended  by  Pasteur  in  1882  has  been  followed.  This 
is  described  by  Pasteur  himself  in  the  following  words  : 

"  With  a  single  lung  we  may  procure  sufficient  virus  to 
serve  for  numerous  series  of  animals.  And  without  hav- 
ing recourse  to  other  lungs  this  provision  may  be  main- 
tained in  the  following  manner  :  It  is  sufficient  before  the 
supply  of  virus  is  exhausted  to  inoculate  a  young  calf  in 
the  dewlap  or  in  the  shoulder.  The  animal  dies  very 
promptly,  and  all  its  tissues  near  the  point  of  inoculation 
are  infiltrated  with  serum,  which  is  virulent,  and  may  be 
collected  and  preserved  in  a  state  of  purity." 

Loir  prefers  to  obtain  the  virus  in  this  way  from  a  calf 
six  to  twelve  months  old,  during  the  second  week  after 
inoculation,  when  the  temperature  of  the  animal  has  gone 
up  to  40°  to  42°  C,  as  the  virus  is  then  said  to  possess  the 
maximum  degree  of  intensity.  This  vaccine  seems  to  be- 
come attenuated  in  passing  through  a  series  of  animals 
by  inoculation,  so  that  when  it  has  been  passed  through  a 
series  of  five  animals  it  no  longer  produces  death  even 
when  inoculated  in  the  most  dangerous  localities.  Loir 
testifies  to  the  protective  value  of  inoculations  with  this 
virus  made  in  the  tail  of  the  animal,  and  gives  the  follow- 
ing example  :  A  few  months  prior  to  the  publication  of 
his  paper  (1893),  about  two  thousand  cows  were  inocu- 


214  IMMUNITY   AND   SERUM-THERAPr. 

lated  with  a  virus  wliicli  had  been  passed  through  a  series 
of  five  calves.  At  the  moment  of  being-  driven  away  they 
were  joined  by  nineteen  other  cows  not  vaccinated.  After 
being  on  the  road  for  a  distance  of  two  thousand  kihj- 
metres  the  animals  arrived  at  their  destination.  The  two 
thousand  vaccinated  were  in  good  condition,  while  eight 
of  the  non-vaccinated  had  died  of  pleuro-pneumonia. 

In  the  BiiUetln  of  Ihe  Central  Sccieti/  of  Veterinary 
Medicine  of  May  24,  1894,  M.  Robcis  reports  the  results 
of  inoculations  made  with  cultures  of  Arloing's  Pneumoha- 
cillus  liquefaciens  bovis,  and  with  injections  of  pulmonary 
serum.  His  statistics  with  reference  to  the  last-mentioned 
"  legal "  inoculations  he  has  obtained  from  official  docu- 
ments relating  to  the  Department  of  the  Seine. 

The  total  number  of  infected  localities  in  this  depart- 
ment during  the  years  1885  to  1891  was  1,253  ;  total  num- 
ber of  contaminated  animals,  18,356  ;  total  number  inocu- 
lated, 18,359  ;  total  number  of  deaths  prior  to  inoculation, 
1,753 ;  total  number  of  deaths  after  inoculation,  2,741  ; 
total  number  of  deaths  due  to  the  inoculation,  94 ;  total 
percentage  of  mortality,  22.8  per  cent.  After  discussing 
these  and  other  statistics  Robcis  arrives  at  the  conclusion 
that  Arloing's  method  of  preventive  inoculations  with 
cultures  of  the  PnetitnohaciUns  Uquefaviens  bo  vis  gives 
better  results  than  the  legal  method  with  serum  from  an 
infected  animal,  the  total  loss  among  animals  exposed  to 
contagion  not  being  over  twelve  to  fourteen  per  cent. 

Nocard  (1892)  says  that  serum  from  the  lungs  of  an 
animal  dead  from  pleuro-pneumonia  preserves  its  viru- 
lence and  usefulness  as  a  vaccine,  when  mixed  with  half  a 
volume  of  pure  neutral  glyceiin  and  half  a  volume  of  a  five 
per  cent,  solution  of  carbolic  acid.  At  the  end  of  two  and 
a  half  months  this  mixture  preserved  its  full  virulence. 


PLEURO-PNEUMONIA   OF   CATTLE.  215 


BIBLIOGRAPHY. 

Arloing,  S.  :   Sur  les  proprietes  putliogenes  des  mati^res  solubles  fa- 

briquees  par  le  microbe  de  la  peripneumonie  contagieuse  des  bovides, 

etc.     Coinpte-reudu,  Acad.  desSci.,  cxvi.,  p.  166,  1893. 
Production  experimentale  de  la  peripneumonie  contagieuse  du 

boeuf  a  I'aide  de  cultures.     Recueil  de  Med.  veterin.,  1894,  p.  505. 
Nocard  :    Moyen  simple  de  conservation  du  virus   peripiieumonique. 

Bull,  de  la  Soc.  Centrale  de  Med.  veter.,  xlvi.,  303,  1893. 
Rebels,  M.  :  Avantage  de  I'inoculalion  preventive,  facultative,  centre 

la  peripneumonie  contagieuse  du  boeuf.     Bull,  de  la  Soc.  Cent,  de 

Med.  veter.,  1894  (May  34th). 
Semmer,  E.  :  Ueber  des  Rinderpestcontagium  und  iiber  Immuuisierung 

und  Schutzimpfung  gegen  Rinderpest.    Berl.  tierarztl.    Wchschr., 

1893,  p.  590. 
Willeras  :  La  vaccination  bovine.     Bull,  de  I'Acad.  R.  de  Med.  de  Bel- 

gique,  1893,  p.  382. 


xin. 

PNEUMONIA. 

The  micrococcus  of  cronpus  pneuftionia  was  discov- 
ered by  the  present  writer  in  the  blood  of  rabbits  inocu- 
lated subcutaneously  with  his  own  saliva  in  September, 
1880.  In  1885  this  micrococcus,  which  I  had  repeatedly 
obtained  in  pure  cultures  from  the  blood  of  rabbits  in- 
oculated, as  in  the  first  instance,  with  my  own  saliva,  was 
identified  with  the  micrococcus  of  the  same  form  present 
in  the  rusty  sputum  of  patients  with  pneumonia.  In  a 
paper  read  before  the  Patholog-ical  Society  of  Philadel- 
phia, in  April,  1885,  and  published  in  the  American  Jour- 
nal  of  the  Medical  Sciences  on  July  1st  of  the  same  year, 
I  say  : 

"  It  seems  probable  that  this  micrococcus  is  concerned 
in  the  etiology  of  croupous  pneumonia,  and  that  the  in- 
fectious nature  of  the  disease  is  due  to  its  presence  in 
the  fibrinous  exudate  into  the  pulmonary  alveoli." 

This  has  since  been  fully  established  by  the  researches 
of  Frankel,  Weichselbaum,  Netter,  Gameleia,  and  many 
others.  Frankel  first  discovered  this  micrococcus  in  his 
own  salivary  secretions  in  1883,  and  his  first  paper  relat- 
ing to  its  presence  in  the  exudate  of  croupous  pneu- 
monia was  published  on  July  13,  1885,  i.e.,  thirteen  days 
after  the  publication  of  the  paper  from  which  the  above 
quotation    is    made.      Under    these    circumstances    the 


PNEUMONIA.  217 

writer  feels  justified  in  ag-ain  calling-  attention  to  his 
priority  in  the  discovery  of  this  important  pathog-enic 
micrococcus,  and  in  objecting-  to  its  being-  described  as 
"  Frankel's  pneumococcus,"  the  "  diplococcus  of  Fran- 
kel,"  etc. 

In  my  iJaper  above  referred  to  (July,  1885)  I  described 
this  micrococcus  under  the  name  of  Micrococcus  Pas- 
teuri,  but  in  my  "  Manual  of  Bacteriology  "  (1892)  it  is 
described  under  the  name  of  Micrococcus  pneumonice 
croiiposce. 

This  micrococcus  is  very  pathogenic  for  mice  and  for 
rabbits,  less  so  for  guinea-pigs  and  for  dogs.  Like  other 
pathogenic  microorganisms  of  the  same  class  it  varies 
greatly  in  virulence  when  obtained  from  different  sources. 
In  the  saliva  of  healthy  persons,  which  seems  to  be  its 
normal  habitat,  it  sometimes  has  comparatively  little 
virulence.  On  the  other  hand,  when  contained  in  the 
blood  or  in  an  exudate  from  a  serous  cavity  of  an  infected 
rabbit  or  mouse,  it  is  very  virulent.  In  one  instance 
(1881)  the  writer  has  seen  a  fatal  result  in  a  dog  from  the 
subcutaneous  injection  of  1  c.c.  of  bloody  serum  from  the 
subcutaneous  connective  tissue  of  a  rabbit  recently  dead. 

Pneumonia  never  results  from  subcutaneous  injections 
into  susceptible  animals,  but  injections  through  the 
thoracic  walls  into  the  lung  may  induce  a  typical  fibrin- 
ous pneumonia.  This  was  first  demonstrated  by  Tala- 
mon  (1883),  who  injected  the  fibrinous  exudate  of  croup- 
ous pneumonia,  obtained  after  death,  or  drawn  during 
life  by  means  of  a  Pravaz  syringe,  from  the  hepatized 
portion  of  the  lung,  into  the  lungs  of  rabbits.  Gameleia 
has  also  induced  pneumonia  in  a  large  number  of  rabbits, 
and  also  in  dogs  and  sheep,  by  injections  directly  into 
the    iDulmonary   tissue.     Sheep    were   found    to    survive 


218  IMMUNITY   AND   SERUM-THERAPY. 

subcutaneous  inoculations,  unless  very  large  doses  (5  c.c.) 
of  a  virulent  culture  were  injected.  But  intrai^ulmonary 
inoculations  are  said  to  have  invariably  produced  a  typi- 
cal fibrinous  pneumonia  which  usually  proved  fatal.  In 
dogs  similar  injections  gave  rise  to  a  "  frank,  fibrinous 
pneumonia  which  rarely  proved  fatal,  recovery  usually 
occurring  in  from  ten  to  fifteen  days,  after  the  animal 
had  passed  through  the  stages  of  red  and  gray  hepatiza- 
tion characteristic  of  this  affection  in  man." 

Without  doubt  an  attack  of  pneumonia  is  followed  by 
a  certain  degree  of  immunity  of  longer  or  shorter  dura- 
tion. According  to  Huge,  who  has  recently  made  a  care- 
ful study  of  the  subject,  relapses  are  very  infrequent — in- 
dicating a  temporary  immunity — but  subsequent  attacks 
are  more  likely  to  occur  in  those  who  have  once  suffered 
an  attack  of  the  disease,  and  as  many  as  four  or  five  at- 
tacks have  been  known  to  occur  in  the  same  individual. 

In  1,100  cases  collected  by  Wagner  but  two  relapses 
occurred  (=  0.18  per  cent.).  Huge  reports  that  in  440 
cases  treated  at  the  Charite  in  Berlin  there  were  but  two 
relapses.  The  liability  to  subsequent  attacks  at  a  later 
period  is  shown  by  the  following  figures,  which  we  copy 
from  Kuge's  paper:  In  280  cases  reported  by  Stortz, 
26.4  per  cent,  had  previously  suffered  an  attack  of  the 
disease ;  in  133  cases  reported  by  Morhart  the  jaropor- 
tion  of  previous  attacks  was  41.3  per  cent.  ;  in  157  cases 
by  Pohlmann,  84.4  per  cent. ;  in  166  cases  by  Schapira, 
31.3  per  cent. ;  in  128  cases  by  Keller,  36.9  per  cent.  :  in 
175  cases  by  Grisolle,  30.9  per  cent. 

The  writer,  in  a  series  of  experiments  made  during  the 
winter  of  1880-81,  obtained  experimental  evidence  which 
showed  that  susceptible  animals  (rabbits)  acquire  im- 
munity from  the  pathogenic  action  of  this  micrococcus 


PNEUMONIA.  219 

as  a  result  of  inoculations  with  an  attenuated  virus.  The 
experiments  referred  to  had  as  their  object  the  determi- 
nation of  the  comparative  vahie  of  various  germicidal 
ag-ents,  as  tested  upon  this  micrococcus ;  incidentally  it 
was  found  "  that  a  protective  intiuence  has  been  shown 
to  result  from  the  injection  "  (into  rabbits)  "  of  virus,  the 
virulence  of  which  has  been  modified,  without  being-  en- 
tirely destroyed,  by  the  agent  used  as  a  disinfectant." 
(Quoted  from  the  writer's  report  of  the  experiments  re- 
ferred to,  Johns  Hopkins  University,  "  Studies  from 
Biological  Laboratory,"  Baltimore,  1882.) 

In  1891  G.  and  F.  Klemperer  published  an  important 
memoir  relating  to  the  pathogenic  action  of  this  micro- 
coccus and  the  production  of  immunity  in  susceptible 
animals  by  means  of  filtered  cultures.  In  some  cases 
this  immunity  was  found  to  last  as  long  as  six  months. 
A  curious  fact  developed  in  their  researches  was  that  the 
potency  of  the  substance  contained  in  the  filtered  cult- 
ures was  increased  by  subjecting  these  to  a  temperature 
of  41°  to  42°  C.  for  three  or  four  days,  or  to  a  higher 
temperature  (60°  C.)  for  an  hour  or  two.  When  injected 
into  a  vein  after  being  subjected  to  such  a  temperature, 
immunity  was  complete  at  the  end  of  three  or  four  days  ; 
but  the  same  material,  not  so  heated,  required  larger 
doses  and  a  considerably  longer  time  (fourteen  days)  to 
confer  immunity  upon  a  susceptible  animal.  The  un- 
warmed  material  caused  a  considerable  elevation  of  tem- 
perature, lasting  for  some  days.  The  authors  mentioned 
ccmclude  from  their  investigations  that  the  toxic  sub- 
stance present  in  cultures  of  Micrococcus  pneumoniae 
crouposse  is  a  proteid  substance,  which  they  propose  to 
call  pneumotoxin.  The  substance  produced  in  the  body 
of  an  immune  animal,  as  a  result  of  protective  inocula- 


220  IMMUNITY    AND   SERUM-THEEAPY. 

tions,  upon  wliicli  the  immunity  of  these  animals  de- 
pends, is  also  a  jsroteid,  which  they  call  antipneumotoxiu. 
This  they  isolated  from  the  blood-serum  of  immune  ani- 
mals. By  exx)eriment  the}'  were  able  to  demonstrate 
that  the  blood-serum  containing  this  pr^otective  proteid, 
when  injected  into  other  animals,  rendered  them  im- 
mune ;  and  also  that  it  arrested  the  progress  of  the  in- 
fectious malady  indiiced  by  inoculating  susceptible  ani- 
mals with  virulent  cultures  of  the  micrococcus,  ^'hen 
injected  into  the  circulation  of  an  infected  animal  its 
curative  action  was  manifested  by  a  considerable  reduc- 
tion of  the  body  temperature.  The  toxalbumin  was  ob- 
tained from  filtered  bouillon  cultures  of  a  virulent  va- 
riety of  the  micrococcus  of  pneumonia,  in  the  form  of  an 
amorphous  yellowish-white  powder.  This  was  thrown 
down  from  the  filtered  cultures  by  means  of  alcohol,  and 
again  dissolved  in  water  and  reprecipitated  in  order  to 
purify  it. 

Issaeff  (1893)  as  a  result  of  his  experiments  has  found 
that  the  virulence  of  this  micrococcus  can  be  greatly  in- 
creased by  successive  inoculations  in  the  peritoneal  cav- 
ity of  rabbits,  and  that  after  a  series  of  ten  or  twelve 
such  inoculations  the  blood  of  the  infected  animal  does 
not  coagulate  and  becomes  extremeh'  toxic.  In  order  to 
obtain  the  toxins  from  this  blood  Issaeff  collects  the 
blood  of  three  or  four  animals  just  dead  in  a  sterilized 
vessel,  and  adds  to  this  an  equal  volume  of  sterilized 
water  containing  one  per  cent,  of  glycerin,  made  alka- 
line by  the  addition  of  a  few  drops  of  a  concentrated  solu- 
tion of  bicarbonate  of  soda.  The  mixture  is  sterilized  by 
passing  it  through  a  Chamberland  filter.  This  liquid 
sometimes  kills  rabbits  when  injected  into  the  circiilation 
in  the  proportion  of  one  per  cent,  of  the  weight  of  the 


PNEUMONIA.  221 

animal.  When  heated  to  70°  C.  its  toxic  power  is  consid- 
erably diminished  and  a  temperature  of  100°  C.  neutral- 
izes it  completely. 

Emmerich  (1891)  has  succeeded  in  immunizing'  rabbits 
and  mice  by  the  intravenous  injection  of  a  very  much  di- 
luted but  virulent  culture  of  the  micrococcus.  Other  rab- 
bits and  mice  were  rendered  immune  by  injecting  into 
them  material  obtained  from  rabbits  immunized  with 
diluted  cultures.  The  flesh  of  these  animals  was  rubbed 
up  into  a  pulp,  and  the  juices  obtained  by  pressure 
through  a  piece  of  sterilized  cloth.  The  bloody  juice, 
after  standing  for  twelve  hours  at  a  temperature  of  10° 
C,  was  passed  throug-h  a  Pasteur  Alter  and  then  served 
to  immunize  the  animals  referred  to. 

Belfanti  (1892)  has  succeeded  in  immunizing  rabbits 
against  the  pathogenic  action  of  this  micrococcus  by  in- 
jecting" into  the  circulation  a  filtrate  obtained  from  the 
sputa  of  pneumonia  cases.  The  viscid  sputa  mixed  with 
an  equal  part  of  distilled  water  was  kept  on  ice  for 
twenty-four  hours  and  then  passed  through  a  Chamber- 
land  filter.  Ten  c.c.  of  this  filtrate  was  injected  into  the 
ear  vein  of  rabbits.  Some  of  the  animals  so  treated 
proved  to  be  immune  against  general  infection  when  in- 
oculated with  a  virulent  culture  of  the  micrococcus,  but 
they  had  a  localized  inflammation  and  oedema  about  the 
point  of  inoculation.  After  recovering  from  this  they 
proved  to  be  entirely  refractory  against  subsequent  in- 
oculations. 

Foa  and  Scabia  (1892)  have  reported  success  in  produc- 
ing immunity  with  filtered  cultures,  and  also  with  a  glyc- 
erine extract  from  the  blood  of  an  infected  rabbit.  This, 
after  filtration,  was  injected  subcutaneously  in  doses 
of  2  c.c.  at  intervals  of  five  days.     The  authors  named 


222  IMMUNITY    AND   SERUM-THERAPY. 

have  also  produced  immunity  in  rabbits  by  the  use  of 
"pneumo-protein."  This  is  an  extract  from  the  bacterial 
cells  obtained  by  first  collecting-  these  from  the  surface 
of  a  Chamberland  filter  throug-h  which  the  cultures  have 
been  passed  ;  then  dig-esting-  them  for  three  hours  at  55° 
C  in  a  five  per  cent,  solution  of  g-lycerin.  According-  to 
Foa  and  Scabia  immunity  produced  in  this  way  is  more 
decided  and  of  long-er  duration  than  that  resulting-  from 
the  other  methods  tested  by  them. 

Mosny  (1892)  has  also  made  numerous  experiments 
which  show  that  rabbits  may  be  immunized  by  means  of 
filtered  cultures,  or  by  the  juices  from  the  tissues  of  an 
immune  animal  obtained  by  maceration  and  filtration. 
When  sterilized  cultures  were  employed  the  best  results 
were  obtained  by  first  heating-  very  virulent  cultures  for 
three  hours  at  60°  C.  The  dose  employed  was  10  c.c,  and 
immunity  was  not  established  immediately  bat  required 
a  period  of  at  least  four  days  for  its  development. 

The  blood-serum  of  immune  rabbits  was  not  found  to 
have  any  bactericidal  power,  and  the  micrococcus  of  pneu- 
monia preserved  its  vitality  longer  in  the  blood-serum  of 
immune  rabbits  than  in  that  of  other  animals  of  the  same 
species. 

G.  and  F.  Klemperer  had  previously  reported  that  the 
blood  of  immune  rabbits  does  not  destroy  the  micrococ- 
cus of  pneumonia  or  restrict  its  development. 

Issaeflf  (1893)  also  reports  his  success  in  immuniziug- 
rabbits  by  means  of  sterilized  cultures  or  filtered  blood 
from  infected  animals  recently  dead.  A  single  intrave- 
nous injection  of  10  c.c.  of  filtered  blood,  prepared  as  here- 
tofore indicated  (p.  220)  suflficed  to  confer  immunity.  To 
test  immunity  the  animals  were  subsequently  inoculated 
with  two  to  four  drops  of  virulent  blood  ;  and  to  maintain 


PJTEUMONIA.  223 

it  the  inoculatious  (0.5  c.c.)  were  repeated  every  four 
weeks.  Although  immune  against  infection  these  ani- 
mals are  said  not  to  have  acquired  any  immunity  against 
the  toxins  of  the  micrococcus  of  pneumonia.  Contrary 
to  the  conclusion  reached  by  G.  and  F.  Klemperer,  Is- 
saeff  concludes  from  his  experiments  that  "  rabbits, 
although  completely  refractory  against  pneumonic  infec- 
tion, remain  highly  sensitive  to  the  toxins  of  this  mi- 
crobe. Even  small  doses  of  the  toxins  are  not  neutralized 
in  the  blood  of  vaccinated  animals.  We  are  therefore 
brought  to  the  conclusion  that  the  existence  of  an  anti- 
toxic property  of  the  blood  of  vaccinated  animals  cannot 
be  admitted." 

The  serum  of  immunized  rabbits  was  not  found  by  Issa- 
eff  to  possess  any  bactericidal  power  for  the  micrococcus 
of  pneumonia,  and  no  attenuation  of  virulence  occurred 
as  a  result  of  cultivation  in  this  serum.  But  when  intro- 
duced beneath  the  skin  of  an  immune  rabbit  the  micro- 
coccus quickly  loses  its  virulence.  At  the  end  of  eigh- 
teen hours  it  has  completely  lost  its  pathogenic  power, 
and  cultures  made  in  bouillon  no  longer  have  any  injuri- 
ous effect  upon  rabbits.  This  attenuating  effect  pro- 
duced in  the  body  of  an  immune  animal  is  ascribed  by 
Issaeff  to  the  action  of  phagocytes,  which  are  said  to  be 
very  numerous,  and  in  the  course  of  five  or  six  hours  to 
pick  up  all  of  the  cocci  in  the  vicinity  of  the  point  of 
inoculation.  These  are  not,  however,  immediately  de- 
stroyed in  the  interior  of  the  phagocytes,  but  preserve 
their  vitality  for  nearly  forty-eight  hoiirs,  and  when  intro- 
duced into  bouillon  give  a  culture  which  has  no  longer 
any  pathogenic  virulence. 


224  IMMUNITY    AND   SERUM-THERAPY. 

Serum-therapy. 

G.  and  F.  Klemperer  (1891)-  having-  succeeded  in  im- 
munizing' rabbits  against  infection  by  this  micrococcus 
IDi'oceeded  to  experiment  with  the  blood-serum  of  im- 
mune rabbits  as  a  therapeutic  agent.  They  were  suc- 
cessful in  saving  infected  rabbits  by  the  intravenous 
injection  of  such  blood-serum  in  the  dose  of  8  c.c.  ad- 
ministered as  late  as  twenty-four  hours  after  the  subcu- 
taneous injection  of  a  virulent  culture.  Subcutaneous 
injections  were  less  effective,  and  often  failed  after  an 
interval  of  twenty -four  hours  from  the  time  of  infection, 
but  were  successful  when  g-iven  within  eight  or  ten  hours. 
The  tissue  juices  of  immune  rabbits  also  proved  to  have 
a  curative  power. 

They  then  proceeded  to  test  the  therapeutic  value  of 
immune  rabbit's  serum  on  man  by  injecting,  in  six  cases, 
from  4  to  6  c.c.  beneath  the  skin.  They  report  that  in 
every  case  a  notable  fall  of  temperature  followed  the  in- 
jection. In  two  cases  it  remained  normal,  and  in  two 
again  mounted  to  the  former  point  at  the  end  of  six 
hours.  The  authors  referred  to  recognized  the  fact  that 
further  experiments  would  be  required  to  establish  the 
value  of  the  treatment. 

Mosny  (1892)  was  not  able  to  confirm  the  results  of  G. 
and  F.  Klemperer  as  to  the  therapeutic  value  of  blood- 
serum  or  tissue  juices  of  immune  animals  when  injected 
into  rabbits  after  infection,  even  when  this  was  done 
after  a  very  short  interval. 

On  the  other  hand,  Foa  and  Scabia  (1892)  succeeded  in 
curing  infected  mice  and  rabbits  by  the  subcutaneous  in- 
jection of  blood-serum  from  immune  animals.  But  in  ten 
cases  of  pneumonia  in  man,  treated  with  doses  of  5  to  7 


PNEUMOl^IA.  22o 

c.c.  of  blood-serum  from  immune  rabbits,  their  results 
were  less  favorable  tliau  those  reported  by  the  Klem- 
perers.  The  dose  mentioned  was  repeated  two  or  three 
times  at  intervals  of  two  days.  In  six  of  the  ten  cases  no 
modification  of  the  course  of  the  disease  occurred,  and  the 
crisis  took  place  on  the  ninth  or  tenth  day.  In  the  other 
four  cases  the  crisis  occurred  in  from  twenty-four  to 
forty-eight  hours  after  the  first  injection,  but  it  would  of 
course  not  be  safe  to  conclude  that  this  was  a  result  of 
the  dose  given. 

Emmerich  (1894),  referring  to  the  experiments  made  by 
Klemperer  and  by  Foa,  says  that  the  rabbits  from  which 
they  obtained  their  serum  were  not  completely  immu- 
nized. In  his  own  experiments  such  a  degree  of  immu- 
nity was  acquired  that  the  animals  resisted  doses  of  30  to 
40  c.c.  of  a  virulent  culture.  This  immunity  did  not,  ac- 
cording to  Emmerich,  depend  upon  the  presence  of  an 
antitoxin  in  the  blood  of  the  immune  animal,  but  upon 
the  destruction  of  the  micrococci  when  introduced  into 
the  body  of  the  animal  by  some  bactericidal  substance 
present  in  the  blood.  Emmerich  and  Fowitzky  have 
given  the  results  of  their  experiments  in  serum-therapy 
in  a  paper  published  in  1891.  These  were  not  numerous, 
but  indicated  that  the  blood-serum  of  rabbits  immunized 
by  their  method  has  the  j)ower  of  curing  the  acute  infec- 
tious disease  (septicaemia)  produced  in  susceptible  ani- 
mals (rabbits  and  mice)  by  inoculation  with  a  culture  of 
the  micrococcus  of  croupous  pneumonia.  In  Emmerich's 
last  published  paper  upon  the  subject  he  gives  no  addi- 
tional experimental  evidence  but  says  : 

"  That  the  active  substance  [Heilstoffe)  in  the  serum  of 
immune  animals  will  also  some  time  prove  to  have  an 
15 


226  IMMUNITY   AND   SERUM-TITERAPY. 

ideal  curative  power  for  man  is  beyond  doubt  and  only  a 
question  of  time." 

BIBLIOGRAPHY. 

Arkharow,  J.  :  Recberches  sur  la  guerisou  de  1' infection  pneumoniquc 

cbez  les  lapius,  etc.     Arch,  de  Med.  Experim.  et  d'Anat.  Pathol., 

iv.,  4. 
Belfanti,  S.  :  Sulla  imraunizatione  del  coniglio  per  mezzo  dei  filtrati  di 

spiito  pueumouico.     Riforma  Med. ,  1892,  No.  126. 
Bunzl-Federu  :  Ueber  Immunisierung  und  Heilung  bei    der  Pneumo- 

kokken-Infektiou.     Arch,  t'tir  Hygiene,  1894,  Bd.  XX.,  p.  152. 
Emmerich,  Rudolf  :  Ueber  die  Infection,  Immuuisirung  und  Heilung 

bei  crouposer  Pueumonie.     Zeitschr.  ftir  Hyg.,  XVII.,  167,  1894. 
Emmerich  und  Fowitzky :  Die  ktinatliche  Erzeugung  von  Immunitat 

gegen   croupose  Pneumonic  und  die    Heilung  dieser    Krankheit. 

Miinch.  med.  Wchscbr.,  1891,  No.  32. 
Foa,  P.,  e  Scabia,  E.  :  Sulla  Pueumoproteina.     Gazz.  Med.  di  Torino, 

1892,  p.  421. 
Issaeflf,    B.  :    Contribution  a  I'etude  de  I'immunite  acquise  contre  le 

pneumocoque.     Ann.  de  I'lnst.  Pasteur,  1893,  p.  260. 
Klemperer,  G.  und  F.  :  Versuche  uber  Immuuisirung  und  Heilung  bei 

der  Pneumokokkenlnfektion.     Berl.    klin.  Wchschr.,  1891,  Nos. 

84  and  35. 
Klemperer,  G.  :  Klinischer  Bericht  fiber  einige  Falle  von  specifisch  be- 

haudelter  Pueumonie.     Centralbl.  f.  klin.  Med.,  1892,  p.  40. 
Ruge,  H.  :    Ueber  das  Pneumonie-Recidive.      Charite-Annalen,    Bd. 

XIX.,  1894,  p.  184. 
Pane,  N.  :  Richerche  sull'  immunizzazione  del  conigli  contro  il  bacillo 
•  setticoemico  dello  sputo  mediante  del  batterio  virulento.     Riv.  Clin. 

eTerapeut.,  1892,  p.  641. 
Ripristinamento    della  virulenza  del    diplo-bacillo  pneumonioe 

mediante  il  virus  carbonchioso.     Riforma  Med.,  1893,  p.  147. 
Mosny,  E.  :  Action  sur  le  pneumocoque  du  serum  sanguin  des  lapins 

vaccines  contre  I'infection  pneumonique.     La  Semaine  Med.,  1892, 

p.  98. 
Sur  la  vaccination  contre  I'infection  pneumonique.     Arch,  de 

Med.  exper.  et  d'Anat.  pathol.,  iv.,  195,  1892. 


xiy. 

RINDERPEST. 

The  disease  of  cattle  known  in  Germany  as  rinderpest 
is  due  to  a  bacillus  closely  resembling-  the  bacillus  of 
fowl  cholera  and  of  swine  plague  {Bacillus  septiccemice 
hemorrhaglcce). 

Professor  Semmer,  of  St.  Petersburg,  has  reported 
(1892)  his  success  in  immunizing  cattle  against  this  dis- 
ease. The  virulence  of  cultures  was  attenuated  by  pass- 
ing them  through  guinea-pigs,  or  by  exposure  to  heat, 
and  this  attenuated  virus  was  used  in  protective  inocu- 
lations.    Semmer  says : 

"  By  the  subcutaneous  injection  of  blood-serum  from 
immune  animals  their  susceptibility  to  rinderpest  was 
diminished,  and  such  blood-serum  destroyed  the  '  rinder- 
pest contagium  '  in  one  to  twenty -four  hours." 

BIBLIOGRAPHY. 

Semmer,  E.  :  Ueber  das  Rinderpestcontagium  und  tiber  Iramunisierung 
und  Schutzimpfung  gegen  Rinderpest.  Berl.  tierarztl.  Wochen- 
schr.,  1893,  590. 


XV. 

SMALL-POX. 

Inoculations  with  virus  obtained  from  a  pustule  on  a 
small-pox  patient  were  extensively  iDractised  before  the 
discovery  of  vaccination  by  Jenuer.  These  inoculations 
o-ave  rise  to  a  mild  attack  of  the  disease,  followed  by  im- 
munity which  was  apparently  as  complete  as  that  follow- 
ing a  more  severe  attack  contracted  in  the  usual  way. 
This  method  seems  to  have  been  practised  by  Eastern 
nations  long-  before  it  was  introduced  into  Europe.  It 
was  extensively  employed  in  Turkey  early  in  the  eigh- 
teenth century,  and  was  introduced  into  England  through 
the  influence  of  Lady  Mary  Wortley  Montagu.  No  doubt 
the  mortality  from  small-pox  was  greatly  diminished  by 
these  inoculations,  but  they  were  attended  by  the  disad- 
vantage that  the  disease  was  propagated  by  them,  in- 
asmuch as  inoculated  individuals  became  a  source  of  in- 
fection for  others.  Inoculation  was  still  practised  in 
England  for  some  time  after  the  demonstration  of  the 
protective  value  of  vaccination,  but  in  1840  it  was  pro- 
hibited by  an  act  of  Parliament.  The  usual  course  of 
the  disease  as  a  result  of  inoculation  was  as  follows  :  On 
the  second  day  a  papule  developed  at  the  point  of  inocu- 
lation, which  became  vesicular  on  the  fourth  day,  and 
pustular  on  the  eighth,  at  which  time  the  patient  had  a 
chill,  followed  by  fever  and  tumefaction  of  the  axillary 


SMALL-POX.  229 

glands.  This  was  followed  by  the  ordinary  small-pox 
eruption  as  seen  in  a  mild  case. 

There  is  some  evidence  that  vaccination  as  a  protection 
against  small-pox  was  practised  to  a  limited  extent  prior 
to  the  time  of  Jenner.  Thus  Von  Humboldt  has  stated 
that  it  was  known  at  an  early  period  to  the  Mexicans. 
But  its  introduction  as  a  reliable  method  of  protecting 
against  smallpox  is  due  to  the  patient  researches  of  the 
renowned  English  physician,  whose  attention  was  first 
attracted  to  the  subject  in  1768,  although  it  was  not  until 
1796  that  he  made  his  first  vaccination  in  the  human  sub- 
ject. His  first  public  institution  for  the  practice  of  vac- 
cination was  established  in  1799,  and  the  following  year 
the  practice  was  introduced  into  France,  Germany,  and 
the  United  States. 

Vaccinia  in  the  cow  was  more  frequent  before  the  in- 
troduction of  vaccination  than  at  present,  and  often  pre- 
vailed as  a  veritable  epidemic.  This  was  no  doubt  due 
to  the  greater  prevalence  of  small-pox,  and  also  to  the 
fact  that  milkers,  not  protected  by  vaccination,  by  the 
sores  on  their  hands,  arising  from  contact  with  the  teats 
of  an  infected  cow,  communicated  the  disease  to  other 
animals. 

We  cannot  attempt  to  review  the  evidence  for  and 
against  the  view  that  vaccinia  in  the  cow  is  simply  a 
modified  form  of  small-pox,  rather  than  a  specific  infec- 
tious disease  of  the  bovine  species,  as  has  been  claimed 
by  numerous  physicians  of  prominence.  We  believe, 
however,  that  the  former  view  is  supported  by  experi- 
mental evidence,  as  well  as  by  the  analogy  resulting 
from  recent  additions  to  our  knowledge  relating  to  pro- 
tective inoculations  in  other  infectious  diseases. 

Nor  is  it  necessary  to  devote  any  space  to  the  methods 


230 


IMMUNITY   AND   SERUM-TIIERAPY. 


of  propagating  vaccine  virus,  and  the  operation  of  vac- 
cinating-, with  its  immediate  results.  But  some  account 
of  the  value  and  duration  of  the  immunity  conferred  by 
a  successful  vaccination  seems  desirable. 

In  the  early  days  of  vaccination  it  was  claimed  that  a 
successful  vaccination  would  confer  a  life-long  immunity 
against  small-pox  ;  but  this  has  been  disapproved  by 
ample  experience,  and  it  is  now  generally  recognized 
that  the  immunity  usually  expires  after  a  time,  which 
varies  greatly  in  different  individuals,  and  can  only  be 
determined  for  each  by  a  revaccination  practised  at 
proper  intervals,  or  when  small-pox  is  prevalent.  The 
fact  that  a  single  attack  of  small-pox  is  not  alwa3's  pro- 
tective would  lead  us  to  expect  that  the  immunity  from 
vaccination  would  not  be  absolute,  and  experience  shows 
that  in  every  small-pox  epidemic  a  certain  number  of 
persons  who  have  been  vaccinated  fall  victims  to  the 
disease ;  but  experience  also  shows  that  the  mortality 
among  the  vaccinated  is  very  much  less  than  among  the 
non-vaccinated.  The  London  Board  of  Health,  from 
thirty  returns  sent  to  them — not  selected — has  given  the 
following  table : 


Natural  small-pox  in  the  un- 
protected   

Small-pox  after  small-pox .... 
•  Small-pox  after  vaccination.. 


Number  of 
Cases. 


1,731 

58 

929 


Deaths. 


361 
22 
32 


Percentage  of 
Deaths. 


20.85 

37.92 

3.44 


The  gradual  loss  of  immunity  from  vaccination,  as 
determined  by  revaccination,  is  shown  by  the  following 
figures,  which  are  given  in  a  recent  (1894)  paper  by  Bie- 


SMALL-POX. 


231 


dert.  In  the  year  1889  small-pox  was  introduced  into 
Hag-enau,  and  twenty-one  cases  occurred.  The  physician 
officiall}^  charg-ed  with  that  duty  revaccinated  all  of  the 
sOhool-children  with  the  result  ffiven  below  : 


Age. 

Number  Vaccinated. 

Successful. 

Per  Cent. 

6  to    7 

288 
292 
322 
221 
306 
413 

94 
187 
Wl 
170 
273 
307 

33  0 

7  to    8 

63  8 

8  to    9 

72  5 

9  to  10 

80  0 

10  to  11 

85  8 

11  to  12 

88.6 

We  may  remark  in  connection  with  this  table  that  in 
Germany  vaccination  is  compulsory,  and  all  children  must 
be  vaccinated  before  the  September  of  the  year  follow- 
ing- their  birth.  All  scholars  in  public  or  private  schools 
who  have  not  had  an  attack  of  small-pox  must  be  revac- 
cinated when  twelve  years  old. 

The  experimental  evidence  presented  in  the  section  on 
acquired  immunity  (Part  First,  Section  II.)  sug-g-ests  the 
possibility  that  in  small-pox  and  other  infectious  dis- 
eases, in  which  the  specific  infectious  agent  has  not  yet 
been  demonstrated,  the  immunity  resulting  from  an  at- 
tack— or  in  small-pox  from  vaccination — may  also  be  due 
to  the  presence  of  an  antitoxin  in  the  blood  of  the  im- 
mune individual. 

The  writer  undertook  some  experiments  with  a  view  to 
determine  this  question  in  the  spring  of  1892,  but  owing- 
to  various  causes  has  never  been  able  to  complete  the  in- 
vestigation. The  experiments  made,  however,  indicate 
that  the  blood  of  a  recently  vaccinated  calf  contains  an 
antitoxin  which  neutralizes  the  potency  of  vaccine  virus, 
either  bovine  or  humanized.     The   exjaeriments   are  re- 


232  IMMUIilTY  AND   SERUM-THERAPY. 

corded  in  my  paper  on  the  "  Practical  Results  of  Bacteri- 
ological Researches  "  as  follows  : 

These  experiments  were  made  with  the  kind  assistance 
of  Dr.  Wm.  E.  Griffiths,  of  Brooklyn,  who  has  for  many 
years  been  engaged  in  the  production  of  vaccine  virus, 
and  consequently  is  an  expert  in  the  vaccination  of  calves 
and  in  recognizing  vaccinia  in  these  animals. 

"Upon  visiting  Dr.  Griffiths  and  making  known  to 
him  my  desires,  I  found  him  quite  willing  to  assist  me, 
and  also  that  he  had  a  recently  vaccinated,  and  conse- 
quently immune,  calf  in  his  stable.  This  animal  had 
been  vaccinated  in  numerous  places  upon  the  abdomen 
and  thighs  fourteen  days  previously.  The  vaccination 
was  entirely  successful,  and  a  large  number  of  quills  had 
been  charged  from  the  vesicles  which  formed.  At  the 
time  of  my  visit  for  the  purpose  of  collecting  blood-serum 
from  this  animal,  dry  crusts  still  remained  attached  at  the 
points  where  vaccination  had  been  practised  two  weeks 
previously.  On  the  28tli  of  April  I  collected  blood-serum 
from  a  superficial  vein  in  the  hind  leg  of  this  calf.  This 
blood  was  i^laced  in  an  ice-chest  for  twenty -four  hours,  at 
the  end  of  which  time  the  clear  serum  was  drawn  off  in 
'  Sternberg's  bulbs.'  Four  drops  of  this  serum  were 
placed  in  each  of  two  small,  sterilized,  glass  tubes ;  in 
one  of  these  we  placed  three  quills  charged  with  fresh 
vaccine  lymph  from  a  calf.  At  the  end  of  an  hour  the 
quills  were  removed,  after  carefully  washing  off  in  the 
serum  the  lymjjh  with  which  they  had  been  charged.  In 
the  other  tube  we  mixed  with  the  four  drops  of  blood- 
serum  an  emulsion  made  from  a  fragment  of  a  perfectly 
fresh  vaccine  crust  from  the  arm  of  a  child ;  this  was 
crushed  upon  a  piece  of  glass  and  rubbed  up  with  a  little 
of  the  same  blood-serum.  The  two  tubes  were  now 
placed  in  an  ice-chest  for  twenty-four  hours,  at  the  end 
of  which  time  the  contents  were  used  to  vaccinate  a  calf 
purchased  for  the  purpose.  Dr.  Griffitlis  carefully  shaved 
the  thiffhs  of  this  calf  and  scarified  each  thiffh  in  several 


SMALL-POX.  233 

places,  as  he  is  accustomed  to  do  in  vaccinating-  for  the 
propagation  of  lymph.  The  contents  of  the  tube  con- 
taining- lymph  from  the  quills  was  rubbed  into  the  scari- 
fied places  upon  one  thig-h,  and  .the  contents  of  the  tube 
containing-  the  emulsified  crust  into  the  other.  On  the 
8th  of  May,  nine  days  after  the  vaccination,  the  calf  was 
carefully  examined,  and  it  was  ascertained  that  the  result 
of  the  vaccination  was  entirely  negative. 

"  Evidently  it  was  necessary  to  make  a  control  experi- 
ment before  we  would  be  justified  in  ascribing-  this  neg-a- 
tive  result  to  a  neutralization  of  the  virus  by  some  special 
substance  present  in  the  blood-serum  of  an  immune  calf. 
Possibly  the  blood  of  a  non-immune  calf  might  also, 
after  an  exposure  of  twenty-four  hours,  neutralize  the 
specific  virulence  of  vaccine  lymph.  The  control  experi- 
ment was  made  as  follows  : 

"On  the  9th  day  of  May  we  collected  blood  from  a  vein 
in  the  leg-  of  a  non-immune  (not  vaccinated)  calf ;  tliis 
was  placed  in  the  ice-chest  for  twenty-four  hours,  and  the 
following-  day  clear  serum  was  collected  in  Sternberg's 
bulbs.  Three  quills,  charged  with  fresh  lymph  from  a 
calf,  of  the  same  lot  as  those  used  in  the  previous  experi- 
ment, were  placed  in  four  drops  of  this  blood-serum  in 
each  of  two  small  glass  tubes.  As  in  the  previous  ex- 
periment, the  lymph  was  washed  from  the  quills  at  the 
end  of  an  hour,  and  the  tubes  were  placed  aside  in  the 
ice-chest.  At  the  end  of  twenty-four  hours  the  serum  in 
these  two  tubes  was  used  to  vaccinate  the  same  calf  which 
had  served  for  the  previous  experiment  Several  points 
were  scarified  upon  the  left  thigh  and  upon  the  left  side 
of  the  abdomen,  which  were  carefully  shaved  for  the  pur- 
pose. 

"At  the  same  time  the  animal  was  vaccinated  upon  the 
right  thigh  and  upon  the  right  side  of  the  abdomen  with 
virus  mixed  with  the  blood-serum  from  the  immune  calf. 
This  serum,  collected  in  Sternberg's  bulbs  on  the  28th  of 
April,  had  since  been  kept  in  the  ice-chest.  One  hour  be- 
fore the  vaccination  four  drops  of  this  blood-serum  were 


234  IMMUNITY   AND   SERUM-TIIERAPY. 

mixed  with  one  drop  of  liquid  lymph,  which  had  been  re- 
cently collected  by  Dr.  Griffiths  in  a  cai^illary  tube  from 
a  vaccinated  calf.  At  the  same  time  three  quills  charged 
with  bovine  lymph  were  immersed  in  four  drops  of  the 
same  blood-serum — from  immune  calf.  As  stated,  the 
animal  was  vaccinated  upon  the  right  side  of  the  ab- 
domen and  upon  the  right  thigh  with  this  virus,  which 
had  been  exposed  for  one  hour  to  the  action  of  blood- 
serum  from  an  immune  calf.  The  serum  containing  the 
liquid  lymph  was  rubbed  into  the  scarification  on  the 
right  side  of  the  abdomen,  the  serum  containing  lymph 
from  the  quills  into  the  right  thigh.  On  the  19th  of 
May,  eight  days  after  the  vaccination,  the  animal  was 
carefully  examined  by  Dr.  Griffiths  and  myself,  and  the 
following  results  noted  :  Upon  the  left  thigh  and  left  side 
of  the  abdomen  the  vaccinations — from  quills  in  non- 
immune blood-serum  after  twenty-four  hours'  contact — 
were  entirelj^  successful,  the  scarification  being  sur- 
rounded by  characteristic  vesicles  and  covered  by  char- 
acteristic crusts.  Upon  the  right  thigh — vaccinations 
from  quills  immersed  in  blood-serum  from  immune  calf 
for  one  hour — and  upon  the  right  side  of  abdomen — vac- 
cinations with  liquid  lymph  mixed  with  blood-serum 
from  immune  calf — the  result  was  entirely  negative. 
Several  of  the  scarifications  had  entirely  healed  ;  others 
were  covered  with  a  dry  scab  which  was  easily  detached 
and  under  which  the  scarification  was  healing  without 
any  appearance  of  vesicles  such  as  surrounded  the  scari- 
fications upon  the  left  side."  * 

Later  I  made  a  number  of  experiments  upon  unvacci- 
nated  children  in  two  orphan  asylums  in  Brooklyn,  with 
a  view  to  ascertain  whether  blood-serum  from  an  immune 
calf,  or  from  an  individual  who  had  recently  sufi'ered  an 
attack  of  small-pox,  if  injected  into  th6   subcutaneous 


*  These  results  have  since  been  confirmed  by  Kinyon. 


SMALL-POX.  235 

tissues  at  the  time  of  vaccination,  would  prevent  the  de- 
velopment of  a  characteristic  vaccine  vesicle.  In  these 
experiments  from  1  to  5  c.c.  of  the  serum  supposed  to 
contain  an  antitoxin  of  small-pox  was  injected  near  the 
l)oint  of  vaccination,  or,  in  some  instances,  into  the  other 
arm.  The  result  was  negative,  even  when  serum  was 
used  from  an  individual  who  was  just  convalescent  from 
a  severe  attack  of  small -pox.  But  it  may  be  that  a  dif- 
ferent result  would  have  been  obtained  if  a  larger  quan- 
tity of  blood-serum  had  been  used,  or  if  it  had  been  in- 
jected into  the  circulation  instead  of  into  the  subcuta- 
neous tissues.  More  recent  experiments  by  Kramer  and 
Boyce  (1893)  and  by  Landmann  (1894)  also  show  that 
small  amounts  of  serum  from  immune  calves  (5  to 
10  c.c.)  do  not  prevent  the  development  of  the  vaccine 
vesicle ;  and  that  blood-serum  (25  c.c.)  from  one  who 
had  suffered  a  recent  attack  of  small-pox  did  not  have 
any  noticeable  effect  upon  the  development  of  a  conflu- 
ent case  of  small-pox  in  a  child  five  years  of  age  (Land- 
mann). 

Tliis  line  of  research,  which  appears  to  us  well  worthy  of 
attention,  is  still  oi)en  for  investigators.  Immune  calves 
may  be  obtained  at  any  vaccine  establishment,  and  it  is  a 
simple  matter  to  collect  sterile  blood-serum  in  consider- 
able amounts  from  these  immune  animals.  We  have  been 
anxious  to  see  a  test  made  as  reg-ards  the  possible  thera- 
peutic value  of  such  serum.  It  could  be  injected  into  the 
circulation  of  a  small-pox  patient  through  the  median 
vein,  and  there  is  no  reason  to  suppose  that  there  would 
be  any  danger  in  introducing-  a  considerable  quantity  in 
this  way.  As  the  writer  is  not  likely  to  have  an  oppor- 
tunity to  follow  up  these  experiments  he  takes  this  occa- 
sion for  inviting-  the  attention  of  others,  more  favorably 


236  IMMUNITY   AND   SERUM-THERAPY. 

situated  for  such  an  investigation,  to  this  promising  field 
for  experimental  work.* 

BIBLIOGRAPHY. 

Anche  :  Essai  de  serotherapie  dans  la  variole.  Arch.  clin.  de  Bordeaux, 
1893,  vol.  ii.,  p.  317. 

Kinyon  :  Preliminary  report  on  the  Treatment  of  Variola  by  its  Anti- 
toxin. Abstract  of  Sanitary  Reports,  Marine  Hospital  Service,  vol. 
X.,  p.  31,  1894. 

Kramer  and  Boyce  :  The  Nature  of  Vaccine  Immunity.  British  !Medi- 
cal  Journal,  Xoveniber,  1893. 

Laudmauu  :  Fiuden  sich  Schutzstoffe  in  dem  Blutserum  von  Individuen, 
welche  Variola  bezw.  Vaccine  iiberstanden  haben  ?  Zeitscbr.  ftlr 
Hygiene,  Bd.  XVIII.,  1894,  p.  318. 

Ruete,  A.,  und  Enoch,  C.  :  Ueber  Vaccinereinkulturen  und  fiber  dus 
toxin  "  Vaccinin."     Dtsche.  med.  Wohschr.,  1893,  p.  547. 

Sternberg  :  Practical  Results  of  Bacteriological  Researches.  Transac- 
tions of  the  Association  of  American  Physicians,  1892,  p.  98. 


*  Since  the  above  was  written,  Kinyon  has  reported  two  cases  in  which  blood- 
serum  from  an  immune  calf  was  injected  subcutaneously  in  small-pox  patients 
in  doses  of  15  to  30  c.  c.  with  supposed  good  results  in  one.  • 


XVI. 

SWINE  PLAGUE. 

As  stated  in  the  chapter  on  cholera  in  fowls,  the  bacil- 
lus of  SAvine  plag'ue  {Schwelneseuche,  Loffler  and  Schiitz) 
very  closely  resembles  Pasteur's  microbe  of  fowl  cholera 
and  Koch's  bacillus  of  rabbit  septicaemia,  and  if  not  iden- 
tical with  these  at  least  varies  from  them  so  slightly  in 
its  morphological  and  biological  characters  that  recent 
authors  do  not  feel  justified  in  considering  it  a  distinct 
species.  Koch  first  obtained  his  bacillus  of  rabbit  septi- 
ciemia  by  inoculating  rabbits  with  putrefying  flesh  infu- 
sion. Gaffky  produced  the  same  infectious  disease  in 
rabbits  by  inoculating  them  with  impure  river-water. 
Davaine  had  previously  obtained  similar  results  by  inoc- 
ulating rabbits  with  putrefying  blood.  The  writer  in 
1887  produced  the  same  disease  in  rabbits,  while  in 
Cuba,  by  inoculating  them  with  putrefying  liver  from  a 
yellow  fever  cadaver.  A  similar,  and  possibly  identical, 
bacillus  has  been  found  in  the  blood  of  deer  (Hueppe),  of 
cattle  (Kitt),  and  of  buffalo  (Oreste  -  Armanni)  suffering 
from  a  fatal  infectious  disease.  And  all  of  these  allied 
species  or  varieties  are  included  by  Huepi^e  and  by  the 
present  writer  under  the  single  specific  name  Bacillus 
septiaemife  liemorrhagicce.  The  bacillus  of  the  disease 
known  in  this  country  as  swine  plague,  according  to 
Smith,  agrees  in  all  particulars  with  that  of  the  German 
swine  plague   {Schweineseuche)   described  by  Lofiler  and 


238  IMMUNITY   AXD   SEKUM-THEUAPY. 

Schiitz,  except  that  tlie  latter  is  more  pathogenic  for 
swine  and  for  rabbits. 

•  In  a  recent  publication  Smith  and  Moore  (United 
States  Department  of  Ag-riculture,  Bureau  of  Animal  In- 
dustry, Bulletin  No.  6,  1894:)  have  given  an  account  of 
their  experiments  relating  to  immunizing  animals  against 
the  pathogenic  action  of  this  bacillus.  The  bacilli  used 
in  these  experiments  were  sufficiently  virulent  to  kill 
rabbits  in  twenty  hours  when  injected  beneath  the  skin 
of  these  animals  in  doses  of  0.001  c.c.  of  a  fresh  bouillon 
culture.  The  experiments  were  made  upon  young  adult 
rabbits  by  various  methods,  viz.  :  with  sterilized  bouillon 
cultures  ;  with  sterilized  suspensions  of  agar  cultures ; 
with  the  filtrate  of  agar  suspensions  ;  with  defibrinated, 
sterilized  blood  of  infected  rabbits;  with  blood-serum 
from  immune  animals. 

"  A  greater  or  less  degree  of  immunity  was  produced 
in  rabbits  by  sterilized  bouillon  cultures,  sterilized  agar 
suspensions,  sterilized  blood  from  infected  rabbits,  and 
blood-serum  from  immunized  rabbits.  The  sterilized 
blood  of  diseased  rabbits  was  capable  of  producing  im- 
munity, while  the  blood-serum  of  immune  rabbits  pro- 
duced rather  equivocal  results." 

The  different  degrees  of  immunity  which  may  be  ac- 
quired by  rabbits,  as  shown  by  a  subsequent  inoculation 
with  virulent  material,  are  classified  by  Moore  as  follows  : 

1.  No  resistance — acute  sei^ticfemia. 

2.  Slight  resistance — peritonitis. 

3.  Increased  resistance — pleuritis  and  pericarditis  with 
or  without  secondary  laneumonia. 

4.  Higher  degree  of  resistance — pleuritis  and  peritoni- 
tis. 


SWINE   PLAGUE.  239 

5.  Still  greater  resistance  —  irregular  lesions  in  the 
form  of  abscesses,  subcutaneous  and  subperitoneal, 

6.  Nearly  complete  immunity — very  slight  reaction  at 
the  point  of  inoculation. 

Uj5  to  the  present  time  the  bacteriolog-ical  experts  of 
the  Department  of  Ag-riculture  have  not  proposed  to 
make  a  practical  application  of  the  facts  developed  in 
their  experimental  work  in  the  way  of  protecting-  herds 
of  swine  by  means  of  inoculations  with  an  attenuated 
virus,  or  with  sterilized  cultures.  In  the  reijort  on  swine 
plague,  made  by  the  Bureau  of  Animal  Industry,  pub- 
lished in  1891,  the  following  measures  for  arresting  an 
epidemic  are  recommended : 

"  When  the  disease  has  actually  appeared  in  a  herd  the 
question  generally  arises  whether  it  is  worth  while  to 
make  any  attempt  to  save  a  portion  of  the  herd  or  to 
leave  them  to  their  fate.  As  a  rule  it  may  be  stated  that 
it  is  best  to  slaughter  both  healthy  and  diseased  at  once, 
and  give  the  surroundings  sufficient  time  to  rid  them- 
selves of  the  infection  before  fresh  animals  are  brought 
into  them.  If  this  be  not  desirable,  we  should  recom- 
mend the  following  measures  to  be  rigorously  carried  out : 

"  a.  Removal  of  still  healthy  animals  to  uninfected 
grounds  or  pens  as  soon  as  possible. 

"  h.  Destruction  of  all  diseased  animals. 

"  c.  Careful  burial  or  burning  of  carcases. 

"  d.  Repeated  thorough  disinfection  of  the  infected 
premises. 

"  e.  Great  cleanliness  both  as  to  surroundings  and  as 
regards  food." 

In  the  same  report  (1891)  the  following  reference  is 
made  to  protective  inoculations  : 

"  As  regards  swine  i)lague  the  experiments  which  have 
thus  far  been  carried  out  indicate  that  this  disease  may 


240  IMMUNITY    AND   SEIU'M-TIIEUAPY. 

prove  amenable  to  preventive  inoculation.  We  have 
been  able,  by  the  injection  of  both  living-  cultures  and 
those  sterilized  at  a  low  temperature  (58°  C.)  to  make  the 
most  susceptible  animals — rabbits — insusceptible  to  the 
most  virulent  swine-plag'ue  bacteria.  By  two  subcutane- 
ous injections  of  cultures  of  swine-plag'ue  bacteria,  swine 
have  been  made  insusceptible  to  doses  injected  into  the 
circulation  which  proved  fatal  to  control  pigs  within 
twenty-four  hours." 

According  to  Smith  the  experiments  of  Metchnikoff 
(1892),  reported  as  made  with  the  bacillus  of  hog  cholera, 
were  in  fact  made  with  the  bacillus  of  swine  plague  ;  we 
therefore  refer  to  them  here.  These  experiments  showed 
that  rabbits  could  be  easily  immunized  against  the  patho- 
genic action  of  virulent  cultures  by  means  of  blood,  from 
an  infected  animal,  sterilized  by  heat.  Doses  of  1.5  c.c, 
or  more,  were  fatal  to  rabbits  ;  but  smallef  doses,  re- 
peated several  times,  given  either  subcutaneously  or  by 
injection  into  the  circulation,  caused  the  animal  to  be- 
come immune. 

BIBLIOGRAPHY. 

Smith  :  Special  Report  on  the  Cause  and  Prevention  of  Swine  Plague. 
United  States  Department  of  Agriculture,  Bureau  of  Animal  In- 
dustry, Washington,  1891. 

Smith  and  Moore  :  Op.  cit.,  p.  238. 


XVII. 

STREPTOCOCCUS   INFECTION. 

It  is  now  generally  recognized  by  pathologists  that 
erysipelas,  puerperal  fever,  certain  forms  of  diphtheritic 
inflammation  of  the  fauces,  and  certain  acute  abscesses 
are  due  to  infection  by  a  streptoccus  described  by  recent 
authors  under  the  name  of  Streptococcus  pyogenes.  This 
streptococcus,  like  other  pathogenic  microorganisms  of 
the  same  class,  varies  greatly  in  its  pathogenic  power  as 
a  result  of  conditions  relating  to  the  source  of  the  par- 
ticular variety  under  cultivation.  As  obtained  from  a 
case  of  erysipelas  or  puerperal  fever  it  is  extremely  vir- 
ulent, but  when  it  has  led  a  saprophytic  existence  for 
some  time,  or  has  been  cultivated  for  a  considerable  time 
in  the  usual  artificial  culture  media,  its  pathogenic  po- 
tency is  greatly  diminished. 

Mironoff  (1893)  has  made  a  series  of  experiments  with 
a  view  to  determining  whether  rabbits  can  be  immu- 
nized against  the  pathogenic  action  of  this  streptococcus, 
and  has  obtained  successful  results  by  the  following 
method  : 

Vigorous  rabbits,  weighing  2  kilogm.,  were  inoculated 
subcutaneously  with  from  3  to  6  c.c.  of  a  sterilized  bouil- 
lon culture  of  the  streptococcus.  Cultures  three  days  old 
were  employed,  and  these  were  sterilized  for  twenty 
minutes  at  120°  C— the  reason  for  using  so  high  a  tem- 

16 


242  IMMUNITY    AND   SERUM-THERAPy. 

perature  is  not  apparent,  inasmuch  as  this  streptococcus 
is  destroyed  in  a  few  minutes  by  a  temperature  of  60°  C. 
At  the  end  of  ten  to  fifteen  days  "  when  the  animal  has 
fully  recovered,"  a  second  dose  of  from  6  to  12  c.c.  of  a 
culture,  sterilized  in  the  same  waj',  is  injected  beneath 
the  skin.  After  another  interval  of  ten  to  fifteen  days  2 
c.c.  of  a  virulent  non-sterilized  culture  is  injected  subcu- 
taneously,  and  this  is  repeated  with  gradually'  increasing 
doses  (1  to  2  c.c.  more)  at  intervals  of  the  same  period. 
Finally  the  animals  "  support  without  reaction  "  a  dose 
five  times  as  great  as  would  be  required  to  kill  an  animal 
of  the  same  weight  not  immunized.  But  the  author 
adds  that  more  than  half  the  animals  thus  treated  died 
before  the  completion  of  the  immunizing  process.  These 
deaths  resulted  from  local  infectious  processes,  such  as 
peritonitis,  pericarditis,  meningitis,  or  abscesses  formed 
at  the  point  of  inoculation. 

Further  experiments  showed  that  the  blood-serum  of 
animals  immunized  in  this  way  when  injected  into  sus- 
ceptible animals  (rabbits)  in  the  dose  of  1.5  c.c.  per  kilo- 
gm.  of  body-weight  conferred  upon  them  a  certain  de- 
gree of  immunity  against  streptococcus  infection,  and 
with  twice  this  amount  (3  c.c.)  a  very  decided  immunity 
was  produced.  The  blood-serum  of  immune  rabbits  in 
doses  of  3  to  4  c.c.  per  kilogm.  of  body-weight  was 
found  to  exercise  a  curative  power,  and  to  completely  ar- 
rest the  acute  septicaemia  resulting  from  inoculations 
with  a  virulent  culture  of  this  streptococcus,  or  to  cause 
the  disease  to  run  a  chronic  course,  with  formation  of 
abscesses  and  final  recovery. 

In  this  connection  we  may  call  attention  to  the  experi- 
ments of  Emmerich  (1886),  which  show  that  the  fatal 
course  of  anthrax  infection,  in  rabbits,  may  be  arrested  by 


STREPTOCOCCUS   INFECTION".  243 

tlie  subcutaneous  or  intravenous  injection  of  this  strep- 
tococcus. Subsequent  experiments  by  Emmerich  and 
de  Mattei  (1887)  showed  that  eleven  hours  after  such  an 
injection  the  anthrax  bacilli  were  all  dead  and  were  al- 
ready undero'oing  degenerative  changes. 

In  a  recent  communication  (1894)  Emmerich  and  his 
associates  have  reported  numerous  additional  experi- 
ments which  show  that  the  blood-serum  of  a  rabbit  which 
is  suffering-  from  streptococcus  septicaemia  (third  day), 
when  filtered  through  a  Pasteur-Chamberland  filter  to 
remove  all  living  cocci,  may  be  used  with  success  in  ar- 
resting anthrax  infection  in  rabbits.  The  filtered  serum 
was  given  four  hours  after  the  anthrax  infection  in  the 
dose  of  25  c.c.  in  the  peritoneal  cavity  and  15  c.c.  subcu- 
taneously.  This  was  repeated  the  following  day  at  nine 
o'clock  in  the  morning  and  five  o'clock  in  the  evening, 
and  again  on  the  third  day  in  the  morning.  Favorable 
results  were  also  obtained  by  using  in  the  same  way 
blood-serum  from  a  sheep  infected  with  the  streptococ- 
cus. 

Cobbett  (1894)  reports  success  in  immunizing  rabbits 
by  means  of  attenuated  varieties  of  the  streptococcus  or 
by  filtered  cultures.  Also  that  cutaneous  erysipelas,  pro- 
duced by  inoculation,  after  recovery  leaves  the  patient 
immune  from  a  repetition  of  the  local  inflammatory  proc- 
ess as  a  result  of  a  subsequent  inoculation,  and  also  con- 
fers a  general  immunity  against  streptococcus  infection. 
But  this  immunity  is  of  short  duration,  not  lasting  long- 
er than  a  few  weeks.  Inoculation  in  the  ear  of  a  rabbit, 
protected  by  a  previous  inoculation  in  the  same  locality, 
is  followed  by  an  inflammatory  reaction ;  but  this  is  of 
brief  duration  and  has  disappeared  before  the  erysipela- 
tous inflammation  jDroduced  in  a  control  is  well  under  way. 


244  IMMUNITY    AND   .SERUM-THERAPY. 

We  may  also  mention  here  the  experiments  of  Mircoli 
(1894)  with  reference  to  the  immunization  of  rabbits 
against  the  pathogenic  action  of  staphylococci  (aureus  ?) ; 
and  of  Rodet  and  Courmont  (1892),  who  have  studied  the 
chemical  products  of  the  staphylococci  and  have  tested 
their  pathogenic  action  upon  dogs  and  rabbits. 

BIBLIOGRAPHY. 

Cobbelt :  Journal  of  Pathology  and  Bacteriology,  vol.  iii.,  p.  88,  1894. 

Emmerich,  R.  :  Die  Heilung  desMilzbrandesdurchErysipelserum,  etc. 
Miiuch.  med.  Wchscbr.,  1894.  Nos.  28-31,  pp.  549,  576,  594,  620. 

Mironoff  :  Immunisation  de.s  lapins  contre  le  streptocoque,  etc.  Arch, 
de  Med.  experim.  et  d'Anat.  pathol.,  1893,  p.  441. 

Mircoli,  S.  :  Immunizzazione  del  coniglio  contre  la  setticemia  da  startl- 
ococco  piogeno.     Gazz.  d.  Ospit.,  1894,  p.  203. 

Rodet,  A.,  et  Courmont,  J.  :  fitude  experimentale  des  substances  solu- 
bles toxiques  elaborees  par  le  staphylocoque  pyogeue.  Rev.  de 
Med.,  1893,  No.  2. 


xvin. 

SYMPTOMATIC  ANTHRAX. 

This  disease  of  cattle  is  popularly  known  as  "  black 
leg-,"  or  "  quarter  evil,"  and  is  described  by  German 
authors  under  the  name  of  Rauschhrand — French,  "  ehar- 
boii  symptoiiiatiqiLer  The  disease  prevails  during-  the 
summer  months  in  various  parts  of  Europe,  and  to  some 
extent  in  the  United  States.  It  is  characterized  by  the 
appearance  of  irregular,  enlphysematous  swellings  of  the 
subcutaneous  tissues  and  muscles,  especially  over  the 
quarters.  The  muscles  in  the  affected  areas  have  a  dark 
color  and  contain  a  bloody  serum  in  which  the  bacillus 
is  found  to  which  the  disease  is  due.  This  is  an  anaero- 
bic bacillus  which  forms  large  oval  spores. 

The  etiolog}^  of  the  disease  was  first  clearly  established 
by  the  researches  of  Arloing,  Cornevin,  and  Thomas  (1880 
to  1883),  and  subsequent  researches  have  shown  that  im- 
munity may  be  produced  in  susceptible  animals  by  pro- 
tective inoculations. 

The  disease  causes  considerable  losses  among  cattle  in 
certain  sections.  Horses  do  not  contract  it  spontaneously, 
and  when  inoculated  with  a  culture  of  the  bacillus  present 
only  a  limited  local  reaction.  Swine,  dogs,  rabbits,  fowls, 
and  pigeons  have  but  slight  susceptibility.  The  re- 
searches of  the  authors  above  mentioned  have  shown  that 
the  virulence  of  a  culture  is  greatly  increased  by  adding 
to  it  twenty  per  cent,  of  lactic  acid.     The  guinea-pig  is 


246  IMMUNITY    AND   SERUM-THERAPY. 

the  most  susceptible  animal  and  succumbs  in  from 
twenty-four  to  thirty-six  hours  when  inoculated  subcu- 
taneously  with  a  small  quantity  of  a  pure  culture.  Ac- 
cording- to  Kitasato  cultures  in  a  bouillon  made  from  the 
flesh  of  the  guinea-pio:  soon  lose  their  virulence,  while 
cultures  in  solid  media  preserve  their  virulence  for  a 
long-  time.  Cultures  ai'e  readily  attenuated  by  heat,  ac- 
cording- to  the  method  of  Toussaiut  and  Chauveau — ex- 
posure to  a  temperature  of  42°  to  43°  C.  in  the  absence 
of  spores.  The  spores  are  attenuated  by  exposure  for 
several  hours  to  a  temperature  of  80°  to  100°  C.  Arloing, 
Cornevin,  and  Thomas  recommend  for  the  production  of 
immunity  in  cattle  inoculation  with  a  dried  jjowder  of 
the  muscles  of  animals  recently  dead  from  the  disease. 
This  is  attenuated  by  heat.  According  to  Kitt  the  mus- 
cles should  first  be  dried  at  32°  to  35°  C.  and  then  pow- 
dered. Two  "  vaccines  "  are  prepared  from  this  powder — 
a  strong  vaccine  by  exposure  to  a  temperature  of  85°  to 
90°  C.  for  six  hours,  and  a  weaker  vaccine  by  exposure 
for  the  same  time  to  a  temperature  of  100°  to  104°  C. 
(dry  heat).  An  inoculation  is  first  made  with  the  weaker 
vaccine  which  gives  rise  to  a  local  reaction  of  moderate 
intensity.  Later  a  second  inoculation  is  made  with  the 
stronger  vaccine,  after  which  the  animal  is  immune  from 
the  pathogenic  action  of  the  most  virulent  material.  Im- 
munity may  also  be  secured  by  intravenous  injections  ; 
or,  in  guinea-pigs,  by  inoculations  with  cultures  which 
have  become  attenuated  by  being  kept  a  few  days,  or  by 
exposure  to  a  temperature  of  42°  to  43°  C. ;  or  by  inocu- 
lation with  a  very  small  quantity  of  a  pure  culture  ;  or  by 
inoculations  with  filtered  cultures  (Roux  and  Chamber- 
land)  ;  or  with  cultures  sterilized  by  heat  (Kitasato).  A 
non-fatal  and  protective  local  infection  may  also  be  pro- 


SYMPTOMATIC   ANTHRAX.  247 

ducecT  in  cattle  by  inoculatious  with  virulent  material 
made  into  the  extremity  of  the  tail.  Roux  has  claimed 
that  animals  which  have  an  acquired  immunity  against 
symptomatic  anthrax  are  also  immune  against  the  patho- 
genic action  of  the  bacillus  of  malignant  oedema;  but 
Kitasato  was  unable  to  confirm  this. 

Strebel,  in  1885,  published  the  results  of  jorotective  in- 
oculations made  in  Switzerland  in  1884.  The  inoculations 
were  made  in  the  end  of  the  tail  with  two  "  vaccines," 
with  an  interval  between  the  two  of  from  nine  to  fourteen 
days.  The  vaccines  were  prepared  by  exposure  to  heat, 
as  above  recommended  by  Arloing,  Cornevin,  and  Thomas. 
The  most  favorable  season  for  inoculations  was  found  to 
be  the  spring,  and  the  most  favorable  age  of  cattle  for 
inoculation  from  five  months  to  two  years. 

In  seven  Swiss  cantons  2,199  cattle  were  inoculated ; 
1,810  inoculations  were  made  among  animals  which  were 
exposed  in  dangerously  infected  pastures.  Of  these  but 
two  died,  one  two  months  and  the  other  four  months  after 
the  protectivjB  inoculations.  Among  908  inoculated  cat- 
tle, which  were  pastured  with  1,650  others  not  inocu- 
lated, the  mortality  was  0.22  per  cent.,  while  the  loss 
among  the  latter  was  6,1  per  cent.  The  following  year 
(1885),  according  to  Strebel,  the  number  of  inoculations, 
exclusive  of  those  made  in  the  canton  of  Bern,  was  35,000. 
The  losses  among  inoculated  animals  are  reported  as 
having  been  about  five  times  less  than  among  those  not 
protected  in  this  way.  In  the  canton  of  Bern,  in  the 
same  year,  according  to  Hess,  15,137  cattle  were  inocu- 
lated by  thirty-eight  veterinarians — 12,190  of  these  were 
pastured  in  dangerously  infected  pastures.  The  results 
are  said  to  have  been  favorable  to  the  method,  but  the 
abstract  at  hand  does  not  give  the  precise  figures. 


248  IMMUNITY    AND   SI<:UUM-TIIEKAPY. 

In  1887  Kitt  repoi*ted  the  results  of  his  investigations, 
which  were  confirmatory  of  those  previously  published 
by  Aiioing-,  Cornevin,  and  Thomas,  and  also  of  a  new 
method  of  inoculation,  which  presented  the  advantag-e 
that  a  single  inoculation  was  sufficient  to  confer  immu- 
nity. This  was  made  in  the  region  of  the  shoulder  with 
a  vaccine  somewhat  stronger  than  that  emjjloyed  by  the 
French  bacteriologists,  but  which  was  found  to  be  with- 
out danger  for  cattle.  It  produced  only  a  slight  local 
effect.  His  vaccine  was  prepared  by  heating  the  moist- 
ened flesh  of  an  animal  just  dead  from  the  disease  to  85° 
to  90°  C.  for  six  hours.  This  did  not  kill  the  spores 
present,  but  caused  a  sufficient  attenuation  in  their  viru- 
lence, t 

In  a  later  communication  (1888)  Kitt  recommends  that 
the  flesh  of  the  diseased  animal  be  first  dried  and  pulver- 
ized, and  then  subjected  to  a  temperature  of  100°  C.  in 
streaming  steam  for  six  hours,  after  which  it  is  to  be 
again  dried  and  used  for  subcutaneous  inoculations. 
The  dose  is  from  5  to  15  ctgr. 

Roux  (1888)  has  shown  by  experiment  that  sterilized 
cultures  of  the  bacillus,  which  have  been  exposed  to  a 
temperature  of  115°  C,  when  injected  in  doses  of  40  c.c, 
three  times  repeated,  into  the  cavit}'  of  the  abdomen  of 
guinea-pigs,  cause  these  animals  to  be  completely  im- 
mune against  the  most  virulent  material.  Cultures  from 
which  the  bacilli  have  been  separated  b}^  filtration  are 
still  more  active.  And  immunity  could  easily  be  con- 
ferred by  the  subcutaneous  inoculation,  in  guinea-pigs, 
of  1  c.c.  of  the  filtrate  from  the  serum  obtained  from  the 
oedematous  tissues  of  a  diseased  animal. 

Schuhanka  (1888)  has  reported  the  results  of  inocula- 
tions made  in  the  dukedom  of  Salzburg  during  the  j^ear 


SYMPTOMATIC   ANTHRAX.  249 

1887.  In  all  2,596  cattle  were  inoculated  once,  and  2,472 
twice,  with  an  attenuated  virus,  in  forty-seven  different 
parishes.  Most  of  these  were  from  six  months  to  a  year 
old.  No  losses  occurred  as  a  result  of  the  inoculations. 
During-  the  summer  of  1887  the  2,472  cattle  Avhich  had 
been  twice  inoculated  were  associated  in  infected  past- 
ures with  3,561  unprotected  cattle.  The  loss  among-  the 
former  was  8,  =  0.32  per  cent.,  among-  the  latter  it  was 
235,  =  6.31  per  cent. 

Strebel  reports  similar  results,  in  1887,  in  the  canton 
Freiburg-,  where  1,725  cattle  which  had  been  inoculated 
suffered  a  loss  of  0.23  per  cent.,  and  1,945  associated  cat- 
tle a  loss  of  5.28  per  cent. 

Lydtin  (1892)  reports  the  results  of  inoculations  made 
in  five  districts  {Amtsbezirken)  in  Baden  during-  the 
years  1886-91  :  2,797  cattle  were  inoculated  with  a  loss 
of  three  only  as  a  result  of  the  inoculation.  None  of 
the  inoculated  cattle  subsequently  contracted  the  dis- 
ease. 

In  the  Bulletin  of  the  Central  Society  of  Veterhiary  Med- 
icine of  France  (1892)  Guillod  and  Simon  give  the  results 
of  3,500  inoculations  made  since  1884.  The  mortality 
among  cattle  in  the  region  where  these  inoculations  were 
practised  had  been  from  ten  to  twenty  per  cent.,  but  fell 
to  0.5  per  cent,  among  the  inoculated  animals. 

The  authors  last  named  prefer  inoculations  in  the  re- 
gion of  the  shoulder  to  the  plan  first  practised  of  inocu- 
lating in  the  end  of  the  tail.  Strebel  also  (1892)  advo- 
cates this  method,  which  is  quickly  carried  out  and  at- 
tended with  but  little  loss.  According  to  Strebel  the 
loss  among  13,022  inoculated  in  this  way  only  amounted 
to  five,  while  the  loss  among  animals  inoculated  by  the 
old  method  was  twice  as  great. 


250  IMMUNITY    AND   SERUM-THERAPY. 


BIBLIOGRAPHY. 

Arloing,  Cornevin  et  Thomas  :  Le  cliarbou  symptomatique  du  boeuf. 
2me  ed.,  Paris,  1887. 

Moyen  de  confeier  artificielleraent  rimmunite  coutre  le  cbarbou 

symptomatique  ou  bacterien  avec  du  virus  atteuue.     Compte-reudu, 
Acad,  des  Sci.,  Paris,  xcv.,  1883,  p.  189. 

Experiences  publiques  sur  la  vaccination  du  cbarbon  symptoma- 
tique.    Arch,  vet.,  Paris,  vi.,  1881,  p.  731. 

De  I'inoculation  du  charbou  symptomatique  par  rnjection  iutra- 


veineuse,  et  de  I'immunite  conferee  au   veau,  au  mouton  et  a  la 

chevre  par  ce  procede.     Compte-rendu,  Acad,  des  Sci.,  Paris,  xci.. 

1880,  p.  734. 
Guillod  et  Simon  :  Du  charbon  symptomatique  ;  nouveau  precede  d'in- 

oculation.     Bull,  de  la  Soc.  Ceutr.   de  Med.  veterin.,  xlvi.,   333, 

1893. 
Hess:  Bericht  iiber  die  Schutzimpfungeu  gegen  Rauschbrand,  etc.,  im 

Kenton  Bern  wahrend  der  Jahre  1886-88.     Bern,  1889. 
Kitt,     T.  :    Ueber    Rauschbraudschutzimpt'ungen    mit    Reinkulturen. 

Mtsch.  f.  prakt.  Tierheilk.,  1893,  p.  19. 
Lydtin  :  Rauschbrandimpfuugen  in   Baden.     Bad.  tbieriirtzl.  Mittheil- 

uugen,  1893,  Nos.  9  and  10. 
Roux  :  Immuuite  contre  le  charbon  symptomatique,  confere  par  les 

substances  solubles.     Ann.  de  I'lnst.  Pasteur,  t.  ii.,  1888,  p.  49. 
Strebel,  M.  :  Protective  Inoculation  for  Symptomatic  Anthrax.     Veter- 
inary Journal,  1893,  p.  153. 
De  I'inoculation  preservative  du  charbon  symptomatique,  etc. 

Jour,  de  Med.  veter.  et  Zootech.,  1893,  p.  11. 

Rauschbraudsehutzimpfung  in  der  Schultergegend,  bzw.  a.  d. 


Brustwand.  Schweitz.    Archiv  f.  Thierheilk.,  XXXIV.,  526,  1893. 
Woltf  :  Schutzimpfungeu  gegen  Rauschljrand.     Berl.   Arch,  fiir  wis- 
seusch.  uud  prakt.     Thierheilk.,  1888,  p.  191. 


XIX. 

TETANUS. 

The  experiments  of  Kitasato  (1889)  show  that  pure  cult- 
ures of  the  tetanus  bacilhis  injected  into  mice,  rabbits, 
or  guinea-pigs  produce  typical  tetanic  symptoms  and 
death.*  As  the  presence  of  this  bacillus  at  the  seat  of  in- 
jury, in  cases  of  tetanus  in  man,  has  now  been  demon- 
strated by  numerous  observers,  there  is  no  longer  any 
question  that  tetanus  must  be  included  among  the  trau- 
matic infectious  diseases,  and  that  the  bacillus  of  Nico- 
laier  and  of  Kitasato  is  the  specific  infectious  agent. 
Kitasato's  recently  published  experiments  (1890)  show 
that  cultures  of  the  tetanus  bacillus  which  have  been 
sterilized  by  filtration  through  porcelain  produce  the 
same  symptoms,  and  death,  in  the  animals  mentioned,  as 
result  from  inoculation  with  cultures  containing  the  ba- 
cillus. It  is  evident,  therefore,  thtU  death  results  from 
the  action  of  a  toxic  substance  produced  by  the  bacillus. 
This  is  further  shown  by  the  fact  that  the  bacillus  itself 
cannot  be  obtained  in  cultures  from  the  blood  or  organs 
of  an  animal  which  has  succumbed  to  an  experimental  in- 
oculation with  an  unfiltered  culture  ;  but  the  blood  of  an 
animal  killed  by  such  an  inoculation  contains  the  tetanus 
poison,  and  when  injected  into  a  mouse  causes  its  death 
with  tetanic  symptoms. 


252  IMMUNITY    AND   SERDM-THEHAPY. 

When  a  platinum  needle  is  clipiDed  into  a  pure  culture 
of  the  tetanus  bacillus,  and  a  mouse  is  inoculated  with  it 
subcutaneously,  the  animal  invariably  falls  sick  within 
twenty-four  hours  and  dies  of  typical  tetanus  in  two  or 
three  days.  Rats,  g-uinea-pigs,  and  rabbits  are  Itilled  in 
the  same  way  by  someAvhat  larger  quantities — 0.3  to  0.5 
c.c.  (Kitasato).  Pigeons  are  very  slightly  susceptible. 
The  tetanic  symiDtoms  are  first  developed  in  the  vicinity 
of  the  point  of  inoculation  ;  if  the  animal  is  inoculated  in 
the  posterior  portion  of  the  body  the  hind  legs  first  show 
tetanic  contraction,  if  in  the  forepart  of  the  body  the 
muscles  of  the  neck  are  first  affected.  At  the  autopsy 
there  is  a  certain  amount  of  hyperaemia  at  the  point  of 
inoculation,  but  no  pus  is  formed ;  in  inoculations  with 
garden  earth,  or  accidental  inoculations  iii  man,  pus  is 
commonly  found  in  the  vicinity  of  the  inoculation  wound. 
The  various  organs  are  normal  in  appearance.  Kita- 
sato says  that  he  has  not  been  able  to  demonstrate  the 
presence  of  the  bacillus  or  of  spores  in  the  spinal  mar- 
row, the  nerves,  muscles,  spleen,  liver,  lungs,  kidneys,  or 
blood  from  the  heart;  nor  has  he  been  able  to  obtain 
cultures  from  the  various  organs.  In  mice  which  were 
inoculated  at  the  root  of  the  tail  Kitasato  was  able  to 
demonstrate  the  presence  of  the  bacilli  at  the  point  of 
inoculation  by  the  microscopical  examination  of  an  ex- 
cised piece  of  the  tissues  for  eight  to  ten  hours  after  the 
inoculation  ;  later  than  this  they  were  not  found.  In  pus 
from  the  inoculation  wounds  of  men  and  animals  acci- 
dentally infected  the  bacilli  are  present,  but  the  forma- 
tion of  spores  does  not  always  occur.  According  to  Kita- 
sato, the  sooner  death  has  occurred  after  accidental 
inoculation  the  less  likely  are  spores  to  be  found  in  the 
rods,  but  from  pus  in  which  no  spores  are  seen  cultures 


TETANUS.  253 

of  the  bacillus  may  be  obtained  in  which  spores  will  de- 
velop in  the  usual  manner. 

Guinea-pig's  are  even  more  susceptible  to  the  tetanus 
poison  than  mice,  and  rabbits  less  so.  The  amount  of 
filtrate  from  a  slightly  alkaline  bouillon  culture  required 
to  kill  a  mouse  is  extremely  minute — 0.00001  c.c.  (Ki- 
tasato).  The  tetanic  symptoms  are  developed  within 
three  days  ;  if  the  animal  is  not  affected  within  four  days 
it  escajjes  entirely.  The  tetanus  poison  is  destroyed  by 
a  temperature  of  65°  C.  maintained  for  five  minutes,  or 
60°  for  twenty  minutes,  or  55°  for  an  hour  and  a  half ; 
in  the  incubating  oven  at  37°  C.  it  gradually  loses  its 
toxic  potency ;  in  diffuse  daylight,  also,  its  toxic  power 
is  gradually  lost ;  in  a  cool,  dark  place  it  retains  its  orig- 
inal potency  indefinitely  ;  in  direct  sunlight  it  is  com- 
pletely destroyed  in  from  fifteen  to  eighteen  hours  ;  it  is 
not  injured  by  being  largely  diluted  with  distilled  water  ; 
it  is  destroyed  in  an  hour  by  hydrochloric  acid  in  the 
proportion  of  0.55  per  cent.  ;  terchloride  of  iodine  de- 
stroys it  in  the  proportion  of  0.5  per  cent.;  cresol  in  one 
per  cent. — one  hour's  exposure.  In  general  it  is  de- 
stroyed by  acids  and  by  alkalies.  Blood-serum  from 
cattle,  horses,  sheep,  rabbits,  rats,  or  guinea-pigs  does 
not  modify  its  toxic  properties. 

Brieger  (1886)  first  succeeded  in  obtaining  from  impure 
cultures  of  the  tetanus  bacillus  a  crystallizable  toxic  sub- 
stance, called  by  him  tetanin,  which  was  found  to  kill 
small  animals  in  very  minute  doses  and  w^itli  the  charac- 
teristic symptoms  of  tetanus.  More  recentl}^  Kitasato 
and  Weyl  have  obtained  the  same  siibstance,  by  follow- 
ing Brieger's  method,  from  a  pure  culture  of  this  bacil- 
lus. From  a  bouillon  made  from  \\  kilogr.  of  lean  beef, 
with  the  addition  of  25  gm.  of  i^eptone,  they  obtained 


254  IMMUNITY   AND   SERUM-THERAPY. 

1.7118  g-m.  of  hydrochlorate  of  tetaniii.  This  proved 
fatal  to  white  mice  in  six  hours  in  the  dose  of  0.05  gm., 
and  a  dose  of  0.105  gm.  caused  characteristic  tetanic  con- 
vulsions and  death  within  an  hour.  The  bacteriolog-ists 
last  named  also  obtained  from  their  cultures  the  tetano- 
toxin  of  Brieger.  Two  mice  were  inoculated  subcutane- 
ously  with  0.003  gm.  of  this  substance ;  one  died  at  the 
end  of  five  hours  "wdthout  the  development  of  tetanic 
symptoms  ;  the  other  survived.  In  addition  to  these 
substances,  indol,  phenol,  and  butyric  acid  were  demon- 
strated to  be  present  in  cultures  of  the  tetanus  bacillus. 

The  more  recent  researches  of  Brieger  and  Frankel,  and 
of  Kitasato,  show  that  the  toxic  jitomaine  discovered  by 
Brieger  in  1886  is  not  the  substance  to  which  cultures 
of  the  tetanus  bacillus  owe  their  great  and  peculiar  pa- 
thogenic power.  The  distinguished  German  chemist  and 
his  associate  have  succeeded  in  isolating  from  tetanus 
cultures  a  toxalhumhi  which  is  far  more  deadly  than 
tetanin. 

Brieger  and  Colin  in  more  recent  investigations  (1893) 
relating  to  the  toxic  products  of  the  tetanus  bacillus  have 
arrived  at  the  following  results  :  The  cultures  were  made 
in  veal  bouillon  containing  one  per  cent  of  peptone  and 
one-fifth  per  cent,  of  chloride  of  sodium.  Large  quanti- 
ties of  the  cultures  in  this  medium  were  filtered  through 
porcelain  filters.  The  active  substance  was  precipitated 
from  the  filtrate  by  means  of  a  saturated  solution  of  am- 
monium sulphate.  By  adding  this  salt  in  excess  the 
precipitate  is  made  to  rise  to  the  surface  and  is  skimmed 
off  with  a  platinum  spatula.  The  liquid  is  removed  by 
placing  this  upon  porous  porcelain  plates  and  the  crude 
toxin  is  dried  in  a  vacuum.  It  still  contains  6.5  per  cent, 
of  ammonium  sulphate.    The  tetanus  bouillon  after  filtra- 


TETANUS.  255 

tion  is  said  to  be  fatal  to  mice  in  the  dose  of  0.00005  c.c. 
A  litre  of  this  bouillon  gave  about  one  gramme  of  the 
dried  precipitate,  which  produced  characteristic  tetanic 
symptoms  and  death  when  injected  into  mice  in  the  dose  of 
0.0000001  gm.  Kitasato  in  his  experiments  had  previously 
obtained  a  tetanus  bouillon  which  was  five  times  as  toxic 
as  that  used  by  Brieger  and  Cohn  in  their  experiments, 
and  which  killed  mice  in  the  dose  of  0.00001  c.c.  The 
dried  precipitate  obtained  by  Brieg-er  and  Cohn  con- 
tained various  impurities,  including-  a  certain  amount  of 
ammonium  sulphate,  but  was  found  to  kill  susceptible 
animals  in  the  proi)ortion  of  0.0000066  g-m.  per  kilo^m. 
of  body-weight. 

It  was  purified  without  loss  of  toxic  power  by  placing 
it  in  a  dialyzer  in  running  water  for  from  twenty-four  to 
forty-eight  hours,  after  which  it  was  dried  in  vacuo  at  20° 
to  22°  C.  The  purified  toxin  as  thus  obtained  had  a 
slightly  yellowish  color,  and  was  in  the  form  of  transpar- 
ent scales,  which  were  odorless,  tasted  like  gum  acacia, 
and  were  easily  soluble  in  water.  The  chemical  reactions 
of  this  purified  toxin,  according  to  Brieger  and  Cohn, 
show  that  it  is  not  a  true  albuminous  body.  When  in- 
jected beneath  the  skin  of  a  mouse  weighing  15  gm.,  in 
the  dose  of  0.00000005  gm.,  it  causes  its  death,  and  one- 
fifth  of  this  amount  gave  rise  to  tetanic  symptoms  from 
which  the  animal  recovered  after  a  time.  The  lethal  dose 
for  a  man  weighing  70  kilogm.  is  estimated  by  the  authors 
named  to  be  0.00023  gm.  (0.23  milligm.).  Comparing  this 
with  the  most  deadly  vegetable  alkaloids  known  it  is 
nearly  six  hundred  times  as  potent  as  atropin  and  one 
hundred  and  fifty  times  as  potent  as  strychnin. 

Fermi  and  Pernossi  (1894),  as  a  result  of  an  elaborate 
research,  have  determined  many  of  the  chemical  charac- 


256  IMMUNITY    AND   SERUM-THERAPY. 

ters  of  tlie  tetanus  toxin.  When  in  solution  it  is  de- 
stroyed by  a  comparatively  low  temperature  (55°  C.  for 
one  hour)  and  by  exposure  to  direct  sunlight,  but  the 
dry  powder  resists  a  temperature  of  120°  C.  It  has  not 
the  properties  of  an  alkaloid,  as  it  is  not  dissolved  by  any 
of  the  usual  solvents  of  these  bodies — the  only  solvent 
thus  far  discovered  is  said  to  be  water.  It  resembles  the 
albumins  and  peptones  in  its  failure  to  pass  throug-h  a 
dialyzing  membrane.  The  authors  last  referred  to  con- 
clude their  summary  of  results  as  follows  : 

"-The  appended  table  shows  that  the  tetanus  poison, 
like  that  of  diphtheria,  in  its  behavior  as  regards  the 
action  of  light,  heat,  chemical  ag-ents,  and  dialysis,  as  also 
its  solvents,  the  ag^ents  which  precipitate  it,  and  its  action 
upon  living-  animals,  closely  resembles  the  poisons  of  ser- 
pents (Naja  tripudians,  Crotalus,  etc.).  As  to  the  chemi- 
cal nature  of  this  group  of  substances  we  can  at  present 
only  say  that  they  rather  have  the  characters  of  colloidal 
substances  than  otherwise,  and  more  nearly  resemble  the 
albuminoid  bodies  than  the  bases.  •  We  do  not,  however, 
reject  the  verj'^  probable  hypothesis  that  these  toxins  are 
acids  or  bases,  or  other  very  unstable,  peculiar  substances 
which  are  closely  united  with  colloidal  substances,  as  is 
the  case,  for  example,  with  the  alkali  and  acid  albumins 
and  so  many  other  albuminous  bodies." 

While  the  exact  nature  of  the  toxic  substance  contained 
in  tetanus  cultures  has  not  been  determined  we  probably' 
cannot,  at  present,  do  better  than  to  continue  to  speak  of 
it  as  a  "  toxalbumin." 

Kitasato  (1891)  was  not  able  to  produce  immunity  in 
mice  by  inoculations  with  minute  doses  of  the  poison,  or 
with  a  filtrate  which  had  been  exposed  to  various  degrees 
of  temperature  by  which  its  activity  was  diminished  or 


TETANUS.  2r>7 

destroyed.  But  immunity  lasting-  for  about  two  months 
was  produced  in  rabbits  by  inoculating  tliem  with  the 
filtrate  from  a  culture  of  the  tetanus  bacillus,  and  subse- 
quently, in  the  same  locality,  with  3  c.c.  of  a  one  jjer  cent, 
solution  of  terchloride  of  iodine  ;  this  last  solution  Avas 
injected  subcutaneously  in  the  same  dose  at.  intervals  of 
twenty-four  hours  for  five  days.  Of  fifteen  rabbits  treated 
in  this  way  six  proved  to  be  immune  against  large  doses 
of  a  virulent  culture  of  the  tetanus  bacillus.  The  same 
treatment  was  not  successful  in  producing  immunity  in 
mice  or  guinea-pigs,  but  the  important  discovery  was 
made  that  a  small  quantity  of  blood  (0.2  c.c.)  from  an  im- 
mune rabbit,  when  injected  into  the  abdominal  cavity  of  a 
mouse,  gave  it  immunity  from  the  effects  of  inoculations 
with  the  tetanus  bacillus.  Moreover,  mice  which  were 
first  inoculated  with  a  virulent  culture  of  the  bacillus, 
and,  after  tetanic  symptoms  had  appeared,  received  in 
the  cavity  of  the  abdomen  an  injection  of  blood-serum 
from  an  immune  mouse,  were  preserved  from  death.  The 
power  of  the  blood  of  an  immune  animal  to  neutralize 
the  tetanus  poison  was  further  shown  b^^  mixing  the  fil- 
trate from  a  virulent  culture  with  blood-serum  from  an 
immune  animal  and  allowing  it  to  stand  for  twenty-four 
hours  ;  a  dose  three  hundred  times  greater  than  would 
have  sufficed  to  kill  a  mouse  proved  to  be  without  effect 
after  such  admixture  with  blood-serum  ;  as  before  stated, 
the  blood-serum  of  animals  Avhicli  are  not  immune  has  no 
effect  upon  the  poison.  The  duration  of  immunity  induced 
in  this  way  was  from  forty  to  fifty  days.  Blood-serum 
from  an  immune  rabbit,  preserved  in  a  cool,  dark  room, 
retains  its  power  of  neutralizing  the  tetanus  poison  for 
about  a  week,  after  which  time  it  gradually  loses  it. 
Having  found  that  chickens  have  a  natural  immunity 
17 


258  IMMUNITY   AND   SERUM-THERAPY. 

against  tetanus,  Kitasato  made  experiments  to  ascertain 
whether  their  blood-serum  would  also  neutralize  the 
tetanus  poison  ;  the  result  was  neg-ative. 

That  the  tetanus  poison  is  present  in  the  blood  of  in- 
dividuals who  die  from  tetanus  has  been  jjroved  by 
Kitasato  by  .injecting  a  small  quantity  (0.2  to  0.3  c.c.)  of 
blood  from  the  heart  of  a  fresh  cadaver  into  mice  ;  the 
animals  develop  typical  tetanic  symptoms  and  die  in 
from  twenty  hours  to  three  days. 

Tizzoni  and  Cattani  have  (1891)  reported  results  simi- 
lar to  those  obtained  by  Kitasato.  By  reiieated  inocula- 
tions with  gradually  increasing  doses  of  the  tetanus 
poison  they  succeeded  in  making  a  dog  and  two  pigeons 
immune,  and  found  that  blood-serum  from  this  immune 
dog,  in  very  small  amount,  completely  destroyed  the 
toxic  power  of  a  filtrate  from  cultures  of  the  tetanus 
bacillus — one  to  two  drops  of  serum  neutralized  0.5  c.c. 
of  filtrate  after  fifteen  to  twenty  minutes'  contact.  They 
also  ascertained  that  small  amounts  of  blood-serum  from 
this  immune  dog  injected  into  other  dogs  or  white  mice 
j)rodiiced  immunity  in  these  animals  ;  but  the}'  were  not 
able  to  produce  immunity  in  guinea-pigs  or  rabbits  by 
the  same  method. 

In  a  later  communication  (May,  1891)  Tizzoni  and  Cat- 
tani give  an  account  of  their  experiments  made  with  a 
view  to  determining  the  nature  of  the  substance  in  the 
blood-serum  of  an  immune  animal  which  has  the  power 
of  destroying  the  toxalbumin  of  tetanus — "  tetanus  anti- 
toxin." They  found,  in  the  first  place,  that  this  antitoxin 
in  blood-senim  is  destroyed  in  half  an  hour  by  a  tem- 
perature of  68°  C  ;  further,  that  it  does  not  pass  through 
a  dialyzing  membrane  ;  that  it  is  destroyed  by  acids  and 
alkalies.     As  a  result  of  their  researches  thev  conclude 


TETANUS.  259 

that  it  is  an  albuminous  substance  having  the  nature  of 
an  enzyme. 

Vaillard  has  succeeded  in  producing  immunity  in  rab- 
bits by  repeated  injections  into  the  circulation  of  filtered 
cultures — in  all  20  c.c. — which  had  been  exposed  for  one 
hour  to  a  temperature  of  60°  C.  At  a  temperature  of 
65°  C.  both  the  toxic  and  the  immunizing-  action  is  de- 
stroyed. 

Behring  (1892)  gives  the  following  account  of  a  method 
which  he  has  successfully  employed  for  producing  im- 
munity in  large  animals — especially  in  horses :  A  culture 
of  the  tetanus  bacillus  is  made,  in  bouillon,  of  such  toxic 
potency  that  0.75  c.c.  will  kill  a  rabbit  in  three  or  four 
days.  To  200  c.c.  of  this  culture  he  adds  carbolic  acid  in 
the  proportion  of  0.5  per  cent,  for  the  purpose  of  preserv- 
ing it.  The  horse  first  receives  a  subcutaneous  injection 
of  10  c.c.  of  this  culture  fluid  to  which  terchloride  of  io- 
dine (I  CI3)  has  been  added  in  the  proportion  of  0.25  per 
cent.  ;  at  the  end  of  eight  days  20  c.c.  of  the  same  mixt- 
ure are  given  ;  again  in  eight  days  the  dose  is  repeated ; 
then,  after  an  interval  of  three  days,  30  c.c.  of  the  same 
mixture.  Following  this,  at  an  interval  of  eight  days,  he 
gives  two  injections  of  30  c.c.  each  of  a  mixture  contain- 
ing one-half  the  quantity  of  I  CI,  (0.175  per  cent.).  The 
proportion  of  the  iodine  terchloride  is  then  reduced  to 
0.125  per  cent.,  and  two  doses  of  20  c.c.  each  are  given. 
Finally  the  culture-fluid  is  administered  in  the  dose  of 
0.5  c.c,  and  this  dose  is  doubled  every  five  days.  Before 
giving  the  first  dose  of  culture-fluid  without  the  addition 
of  I  CI3,  the  immunizing  value  of  the  blood-serum  of  the 
horse  is  tested  upon  mice,  and  if  it  falls  below  1  to  100  a 
dose  of  0.25  c.c.  is  given  instead  of  the  larger  dose  (0.5 
c.c.)  above  mentioned. 


260  IMMUNITY   AND    SERUM-THERAPY. 

Schiitz  (1892)  lias  applied  Beliring's  method  to  a  con- 
siderable number  of  horses  and  sheep,  and  arrives  at  the 
conclusion  that  it  is  a  reliable  method  of  protecting  these 
animals  against  infection  with  living  tetanus  bacilli  and 
against  the  toxic  action  of  filtered  cultures  ;  that  the  de- 
gree of  immunity  and  the  antitoxic  j)ower  of  the  blood- 
serum  increases  as  larger  doses  are  gradually  given.  Ac- 
cording to  Behring  the  immunizing  value  of  blood-serum 
from  a  horse  treated  in  this  way  is  very  high.  As  tested 
on  mice  it  may  be  1  to  200,000,  or  even  more.  According 
to  his  calculations  a  serum  having  a  value  of  1  to  100,000, 
as  tested  on  mice,  should  be  given  to  a  man  weighing  50 
kilogr.  in  the  quantity  of  50  c.c,  given  in  the  course  of 
two  days,  in  order  to  insure  immunity. 

The  same  author  in  a  subsequent  paper  (1892)  gives 
details  as  to  the  method  of  estimating  the  therapeutic 
value  of  serum  from  an  immune  animal.  He  first  calls 
attention  to  the  fact  that  the  only  reagent  by  which  the 
antitoxic  potency  of  this  serum  can  be  tested  is  the  body 
of  a  living  animal.  The  test  animal  selected  is  the  white 
mouse.  When  the  statement  is  made  that  a  serum  has 
the  value  of  1  to  1,000,000,  he  means  that  by  an  experi- 
mental test,  made  upon  white  mice,  it  has  been  ascer- 
tained that  these  animals  are  protected  from  fatal  infec- 
tion with  the  minimal  lethal  dose  of  a  tetanus  culture  by 
the  use  of  0.00002  gm.  of  the  serum  for  a  mouse  weighing 
20  gm.  For  the  cure  of  tetanus  in  the  mouse,  after  the 
first  symptoms  of  the  disease  have  appeared,  a  dose  at 
least  one  thousand  times  as  great  as  the  immunizing 
dose  is  required,  and  the  more  advanced  the  progress  of 
the  case  the  greater  the  dose  must  be.  A  serum  of  the 
strength  above  indicated,  if  used  for  the  treatment  of  a 
case  of  tetanus  in  man,  should,  according  to  Behring,  be 


TETANUS.  261 

employed  in  doses  amounting'  altogether  to  at  least  100 
c.c. — g-iven  inside  of  twenty-four  hours  in  doses  of  20  c.c. 
each.  For  persons  sixteen  years  old  he  would  give  doses 
of  10  c.c,  and  for  children  under  six,  5  c.c.  at  a  dose. 
The  serum  of  this  strength  which  he  had  prepared  for 
testing-  its  curative  value  on  man  was  preserved  by  the 
addition  of  0.5  per  cent,  of  carbolic  acid. 

Rotter  (1892)  reports  a  case  successfully  treated  by 
Behring's  serum.  In  all  250  c.c.  was  administered  sub- 
cutaneously.  The  case  was  not,  however,  one  of  the 
most  severe  forms  of  the  disease. 

Brieg-er  and  Ehrlich  (1892)  have  succeeded  in  immuniz- 
ing' g-oats  by  means  of  gradually  increasing-  doses  of  a 
culture  of  the  tetanus  bacillus  in  thymus  bouillon.  The 
amount  g-iveu  at  first  was  0.2  c.c,  and  this  was  gradually 
increased  to  10  c.c.  At  the  end  of  thirty-seven  days  the 
animal  was  found  to  be  immune  against  virulent  cultures, 
and  the  important  fact  was  demonstrated  that  the  im- 
munizing substance  (antitoxin)  was  present  in  its  milk. 
A  mouse  which  received  0.1  c.c.  of  the  milk  of  this 
goat  in  the  peritoneal  cavity  proved  to  be  immune 
ag-ainst  infection  as  a  result  of  inoculation  with  a  tetanus 
culture.  The  immunizing-  value  of  the  milk  from  this 
goat  was  found  to  be  1,600.  That  is,  a  dose  of  0.2  c.c, 
which  was  equal  to  1  to  109  of  the  body-weig-ht  of  the 
animal,  protected  a  mouse  from  sixteen  times  the  fatal 
dose  of  a  tetanus  culture.  After  precipitation  of  the 
casein  the  milk  still  preserved  its  antitoxic  power  unim- 
paired, and  by  concentrating-  it  in  vacuo  a  fluid  was  ob- 
tained which  proved  to  have  an  immunizing-  value  of 
5,000.      • 

In  a  later  communication  (1893)  Brieger  and  Colin 
give  the  results  of  additional  experiments  with  the  milk 


262  IMMUNIXr   AND   SERUM-THERAPY. 

of  immunized  g-oats.  Animals  were  chosen  which  were 
two  or  three  years  old  and  had  given  birth  to  young-  a 
few  weeks  before  the  inoculations  were  commenced.  It 
having  been  previously  shown  by  Ehrlich  that  the  pre- 
cipitated  tetanus  toxin  from  cultures  could  be  success- 
fully used  to  immunize  guinea-pigs,  the  same  substance 
was  employed  in  these  experiments.  The  treatment 
was  commenced  with  a  dose  of  0.00001  gm.,  which  was 
carefully  increased  to  0.00007  gr.,  the  injections  being 
made  at  intervals  of  four  days.  But  this  proved  to  be 
too  much,  and  the  animal  died  of  typical  tetanus  after 
the  last  dose.  In  a  subsequent  experiment  Brieger  and 
Cohn  succeeded  in  immunizing  a  goat  in  a  month  and  a 
half  so  that  the  animal  finally  withstood  a  dose  of  0.06 
gm.,  but  this  animal  ceased  to  give  milk,  became  anae- 
mic, and  finally  died. 

The  authors  therefore  resorted  to  a  different  method 
which  had  previously  been  successfully  employed  by 
Ehrlich,  Behring,  and  others.  Cultures  of  the  tetanus  ba- 
cillus in  bouillon  were  heated  to  65°  C.  for  half  an  hour, 
and  then  used  for  immunizing  two  goats.  After  five 
weeks'  treatment  the  animals  resisted  doses  of  the  pre- 
cipitated toxin,  which  were  gradually  increased  to  10  gm., 
at  which  time  the  treatment  had  been  carried  on  for 
nearly  six  months  and  the  antitoxic  value  of  the  milk 
was  found  to  be  90,000  immunization  units. 

The  method  of  determining  antitoxic  values  adopted 
by  Brieger  and  Cohn  is  the  following  :  They  had  found 
by  carefully  conducted  experiments  that  their  precipi- 
tated toxin  {Rohgifte)  killed  a  mouse  weighing  20  gm.  in 
the  dose  of  0.0000003  gm.,but  failed  to  kill  when  injected 
in  the  dose  of  0.0000002  gm.  The  first-mentioned  dose 
was  therefore  accepted  as  the  minimum  fatal  dose  for  an 


TETANUS.  "  263 

animal  weig-liing-  18  to  20  g-m.,  and  the  object  in  view  was 
to  find  the  minimum  amount  of  milk  required  to  prevent 
the  toxic  action  of  such  a  dose. 

The  antitoxin  was  obtained  from  the  g-oat's  milk  by 
precipitation  with  ammonium  sulphate,  thirty-two  per 
cent. ;  the  precipitate  was  again  dissolved  and  treated 
with  a  solution  of  basic  acetate  of  lead ;  this  salt  does 
not  precipitate  the  antitoxin  when  the  solution  is  slight- 
ly alkaline ;  the  voluminous  precipitate  produced  by  the 
lead  acetate  is  filtered  out  and  repeatedly  washed  with 
water ;  the  filtered  fluid  and  wash-water  are  again  treated 
with  ammonium  sulphate,  added  to  saturation,  and  the 
resulting  precipitate  is  dissolved  in  a  small  quantity  of 
water  ;  a  precipitate  is  again  obtained  by  saturation  with 
ammonium  sulphate,  and  this  is  dried  upon  porcelain 
plates  in  a  vacuum.  The  ammonium  sulphate  remaining 
could  not  be  removed  by  dialysis,  as  experiment  showed 
that  a  considerable  loss  of  the  antitoxin  occurred  in  a  di- 
al.yzer  placed  in  running  water.  But  by  shaking-  up  the 
dry  powder  in  chloroform  the  heavy  salt  sank  to  the  bot- 
tom and  the  purified  antitoxin  floated  on  the  surface  and 
could  be  recovered  by  skimming  it  off.  The  powder  thus 
obtained  consisted  of  a  mixture  of  various  substances,  in- 
cluding the  antitoxin,  and  wdien  obtained  from  milk  hav- 
ing an  antitoxic  value  of  90,000  it  was  found  to  have  a 
value  of  25,000,000  immunization  units.  By  further  puri- 
fication a  still  higher  value  was  obtained  (55,000,000).  In 
experiments  on  mice  a  dose  ten  thousand  times  as  great 
as  was  necessary  to  produce  immunity  proved  to  exercise 
a  curative  power— i.e.,  a  dose  of  0.02  gm.  for  a  mouse 
weighing  20  gm.  saved  it  from  being  killed  by  double  the 
minimum  fatal  dose  of  the  tetanus  toxin,  after  tetanic 
symptoms  had  been  developed. 


264  IMMUNITY    AND   SERUM-THERAPY. 

Reference  has  been  made  to  the  production  of  immu- 
nity by  the  use  of  cultures  made  in  thymus  bouillon. 
This  was  made  known  through  the  experiments  of  Brie- 
ger,  Kitasato,  and  Wassermann  (1892).  The  thymus 
bouillon  is  made  from  the  thymus  glands  of  calves,  which 
are  chopped  fine  in  a  hash-machine  and  covered  with  an 
equal  volume  of  distilled  water.  The  mixture  is  stirred 
for  some  time  and  then  placed  in  an  ice-chest  for  twelve 
hours  ;  the  liquid  is  then  obtained  by  filtration  through 
gauze  with  pressure — by  means  of  a  flesh-press  machine. 
A  turbid,  slimy  fluid  is  thus  obtained,  which  is  diluted 
with  an  equal  volume  of  water  and  made  slightly  alkaline 
by  the  addition  of  soda  solution.  It  is  then  sterilized  at 
100°  C.  for  fifteen  minutes.  As  a  result  of  this  the  liquid 
has  a  grayish-brown  color,  and  some  large  flocculi  in  sus- 
pension, which  are  removed  by  passing  it  through  fine 
linen.  The  fluid  is  then  of  a  milky  opalescence.  It  is 
next  placed  in  test  tubes  and  again  sterilized.  The  tet- 
anus bacillus  when  cultivated  in  this  medium  does  not 
form  spores,  and  the  toxic  potency  of  the  culture  is  very 
much  reduced — 1  to  5,000  to  1  to  3,000  of  the  toxic  potency 
manifested  by  cultures  of  the  same  bacillus  in  ordinary 
media.  Inoculations  with  cultures  in  thymus  bouillon 
were  found  to  kill  mice  in  the  dose  of  0.5  c.c,  while  small- 
er amounts  failed  to  kill  and  caused  the  animals  to  be 
immune.  A  culture  in  ordinary  bouillon  was  fatal  to 
mice  in  the  dose  of  0.001  c.c. 

Experiments  on  rabbits  (thirty-five)  gave  a  uniformly 
successful  result  in  immunizing  these  animals.  Immu- 
nity was  established  in  the  course  of  two  weeks,  and  the 
blood-serum  of  these  animals  tested  on  mice  showed  an 
antitoxic  value  of  1,000. 

Reference  has   already   been   made  to   the   earlier  re- 


TETANUS.  265 

searches  of  the  Italian  investigators,  Tizzoni  and  Cat- 
tani.     These  have  been  followed  by  additional  investi- 
gations, the  results  of  virhich    have    been    reijorted  in 
numerous  published  iiapers.     The  authors  named  have 
ascertained  that  when  kept  in  a  cool  place  (15°  to  25°  C.) 
the  blood-serum  of  immune  rabbits  retains  its  antitoxic 
power  for  several  months,  and  the  antitoxin,  obtained  by 
j)recipitation  with  alcohol,  kept  in  a  dry  condition  for  more 
than  ten  months,  was  found  to  preserve  its  original  activity. 
Having  succeeded  in  their  earlier  experiments  in  immu- 
nizing rabbits  and  dogs,  Tizzoni  and  Cattani  (in  1893) 
proceeded  to  experiment  upon  horses,  and  were  equally 
successful  with  these  animals.     As  a  result  of  numerous 
injections  with  an  attenuated  virus,  continued  for  a  pe- 
riod of  ninety-seven  days,  they  established  an  immunity 
which  was  tested  by  inoculating  the  animal  with  10  c.c, 
of  a  gelatine  culture,  of  which  l-200th   part  of  a  drop 
killed  a  white  mouse.     The  antitoxic  value  of  the  blood- 
serum  of  this  horse  was  1  to  5,000,000— he.,  1  gm.  of  this 
serum  would  immunize  5,000,000  gm.  of  mice,  or  250,000 
mice  weighing  20  gm.  each.     In  a  later  communication 
(1894)  the  authors  named  report  that  after  freely  bleed- 
ing immunized  horses,  and  allowing  them  to  rest  for  one 
or  two  months,  and  then  again  treating  them  with  small 
doses  of  tetanus  cultures,  the  blood-serum  soon  becomes 
as  active  as  before  the  bleeding.     The  greatest  antitoxic 
power  was  manifested  from  twenty  to  twenty-three  days 
after  the  completion  of  the  protective  inoculations,  and  a 
serum  was  obtained  possessing  a  value  of  1  to  10,000,000. 
According  to    the   authors  named  the  precipitated   (by 
alcohol)  and  purified  antitoxin  from  such  a  serum,  judg- 
ing from  their  experiments  on  lower  animals,  should  cure 
a  case  of  tetanus  in  man  in  the  dose  of  40  to  50  ctgr. 


266  IMMUNITY   AND   SERUM-THERAPY. 

The  authors  last  nientioneLl  have  reported  (1892)  thut 
the  young-  of  immuue  i:)arents  have  a  certain  degree  of  m- 
herited  immunity.  And  the  more  recent  experiments  of 
Ehrlich  and  Hiibener  have  confirmed  this  so  far  as  the 
inheritance  of  immunity  from  the  mother  (in  mice)  is 
concerned  ;  but  their  results  did  not  show  any  immunity 
in  the  young-  when  only  the  father  had  been  rendered 
immune  ;  and  the  immunity  inherited  from  the  mother 
only  lasted  for  two  or  three  months  after  birth. 

Serum-therapy  in  Man. 

The  evidence  has  already  been  presented  in  support  of 
the  therapeutic  value  of  the  blood-serum  from  animals 
immunized  against  tetanus,  and  of  the  precipitated  anti-^ 
toxin  from  such  serum.  It  only  remains  to  present  the 
results  of  the  same  treatment  as  applied  to  tetanus  in 
man.  Dr.  Rudolph  Schwarz,  Assistant  to  the  Surgical 
Clinic  of  Padua,  reported  the  first  successful  case,  treated 
with  an  antitoxin  prepared  by  Professor  Tizzoni  from  the 
blood-serum  of  a  dog  which  he  had  immunized  against 
tetanus.  The  following  is  an  abstract  of  the  case  as  re- 
ported by  Dr.  Schwarz : 

"  The  patient  was  a  peasant  boy,  aged  fifteen  years, 
who  wounded  himself  in  the  left  forearm  while  attempt- 
ing to  cut  a  walnut  which  he  had  picked  up  from  the 
ground.  There  was  considerable  hemorrhage,  which  was 
controlled  by  the  application  of  a  wad  of  spider's  web 
and  a  bandage.  A  return  of  the  hemorrhage  induced  the 
parents  to  take  the  boy  to  the  Surgical  Clinic  at  Padua. 
There,  under  appropriate  treatment,  the  hemorrhage  was 
arrested  and  the  wound  healed.  Two  weeks  after  receiv- 
ing the  wound  symptoms  of  tetanus  were  developed. 
The  patient  was  admitted  to  hospital  three  days  later,  at 


TETANUS.  267 

which  time  the  jaws  were  immovable  and  the  muscles  of 
the  arm  in  a  state  of  tetanic  rigidity,  while  other  muscles 
of  the  trunk  and  extremities  were  also  involved.  Treat- 
ment with  chloral  and  warm  baths  was  without  effect  in 
arresting-  the  progress  of  the  malady,  and  on  the  16th  of 
September,  nine  days  after  admission,  the  patient  had 
eight  or  nine  tetanic  convulsions  during  the  night,  ac- 
companied by  difficult  respiration  and  slight  opisthot- 
onos. No  improvement  having  occurred  under  the 
treatment  employed,  it  was  decided  to  test  the  value  of 
tetanus  antitoxin,  which  had  been  sent  for  the  purpose 
by  Professor  Tizzoni,  who  had  obtained  it  from  the 
blood-serum  of  a  dog  which  had  been  rendered  '  strongly 
immune '  against  tetanus.  On  the  afternoon  of  Septem- 
ber 18th,  15  ctgr.  of  this  antitoxin,  dissolved  in  water, 
were  injected  beneath  the  skin.  The  same  quantitj^  was 
injected  the  following  day.  On  the  morning  of  the  20th 
the  patient  Avas  decidedly  better ;  on  the  afternoon  of 
this  day  he  received  25  ctgr.  of  the  antitoxin,  and  the 
same  amount  on  the  following  day.  After  each  injec- 
tion there  was  a  notable  fall  in  the  temperature.  The 
patient  continued  to  improve,  and  on  the  23d  all  symp- 
toms of  tetanus  had  disappeared." 

In  a  postscript  to  his  communication  Dr.  Schwarz 
states  that  he  has  private  information  of  two  other  pa- 
tients who  have  recovered  from  tetanus  under  the  same 
treatment — one  in  the  hospital  at  Colle  di  Val  d'Elsa 
(Tuscany),  treated  by  Dr.  Pacini  ;  the  other  in  the  Surgi- 
cal Clinic  at  Innsbruch,  treated  by  Professor  Nicoladoni. 

Eenon  (1892)  reports  two  cases  treated  by  subcutaneous 
injections  of  the  blood  of  immune  rabbits  (one  received  in 
all  57  c.c,  the  other  80  c.c).  Both  resulted  fatally,  but 
an  amelioration  of  the  symptoms  was  noted  in  both  cases 
after  each  injection  of  the  antitoxic  serum. 

Baginsky  (1892)  reports  the  case  of  a  child,  nine  days 


268  IMMUNITY    AND   SERUM-THERAPY. 

old,  in  which  tetanus  was  developed  as  a  result  of  infec- 
tion through  the  navel.  The  case  was  treated  by  Kita- 
sato  with  serum  from  an  immune  rabbit  in  doses  of  0.1 
to  0.4  C.C.,  repeated  daily  (total  amount  used  1.5  c.c).  The 
child  died  on  the  thirteenth  day,  and  Baginsky  infers 
that  the  doses  employed  were  too  small. 

Additional  cases  have  been  reported  as  successfully 
treated  by  several  Italian  physicians  (Casali,  Finotti, 
Gagliardi,  Pacini,  Taruffi).  In  all  of  these  cases  the  dry 
antitoxin  supplied  by  Tizzoni  and  Cattani,  and  obtained 
by  them  from  the  serum  of  immunized  dogs,  was  em- 
ployed. The  dry  powder  in  the  dose  of  0.25  gm.  was 
dissolved  in  water  and  injected  subcutaneously.  In  an- 
other case  reported  by  Tizzoni  40  c.c.  of  rabbit  serum 
was  used.  Recovery  occurred  in  all  of  the  cases.  In  one 
very  severe  case  (the  tenth  treated)  reported  by  Finotti, 
improvement  occurred  after  the  administration  of  the 
antitoxin,  but  the  supply  was  exhausted  and  the  tetanic 
symiDtoms  again  became  more  severe  ;  upon  the  receipt 
of  an  additional  supply  and  a  renewal  of  the  treatment 
improvement  again  occurred  and  the  case  recovered. 

The  eleventh  successful  case  is  reported  by  Dr.  Gattai, 
of  Pisa,  the  twelfth  (1893)  by  Lesi,  of  Imola. 

BIBLIOGRAPHY. 

Behring,  E. :  Das  Tetanusheilserum  uud  seine  Anwendung  auf  tetanus- 

kranke  Meuscben.     Leipzig,  1892  (Thieme). 
Ueber  Iramunisirung  uud   Heilung  von   Versuclisthieren  bcim 

Tetanus.     Zeilscbr.  fiir  Hyg.,  XII.,  45,  1893. 
Bebring  und  Knorr :  Ueber  den  Immunisirungswertb  und  Heilwert  des 

Tetanusbeilserums  bei  weissen  Mausen.    Zeitscbr.  fiir  Hyg.,  XIII., 

407,  1893. 
Behring   und   Kitasato  :  Ueber  das  Zustandekommen  der  Dipbtherie- 

Immunitat  und  der  Tetanus-Immunitat.    Deutsche  med.  Wchschr., 

1890,  No.  49. 


TETANUS.  269 

Briegei-und  Colin  :  Untersuchungen  tiber  das  Tetanusgift.     Zeitschr. 

ftlr  Hyg.,  XV.,  1,  1898. 
Beitrage  zur  Konzeiitrierung  gegen  WunSstarrkriimpjI  scliiizen- 

den  substauz  aus  der  jVIilch.     Zeitschr.  f.  Hyg.,  1893,  489. 
Courniont,  J.,  et  Doyon,  M. :  La  substance  toxique  qui  engendre  le  te- 

tanos  resulte  de  Faction  sur  I'orgauisme  recepteur  d'un  ferment 

soluble  fabrique  par  le  bacille  de  Nicolaiier.     Compte-rendu,  8oc. 

de  Biol.,  1893,  p.  394. 
De  la  production  du  tetanos  chez  la  poule,  et  de  la  creation  arti- 

ficielle  de  rimraunite  chez  cet  animal.     Compte-rendu,    Soc.  de 

Biol.,  1893,  p.  841. 
Ehrlich,  P.,  und  Htibeuer,  W.:  Ueber  die  Vererbnng  der  Imniuiiitat 

bei  Tetanus.     Zeitschr.  f.  Hyg.,  XVIH.,  51,  1894. 
Fedoroff,  S.  :  Wirkt  das  Tetanusaiititoxin  auch  giftzerstorend.     Cen- 

tralbl.  f.  Bacteriol.,  XVI.,  484,  1894. 
Fermi,  C,  und  Pernossi,  L. :  Ueber  das  Tetanusgift.     Centralhl.  f.  Bak- 

teriol.,  XV.,  303,  1894. 

Ueber  das  Tetanusgift.     Zeitschr.  ftir  Hyg.,  XVI.,  p.  386,  1894. 

Kitasato :    Experimentelle     Untersuchungen    iiber    das     Tetanusgift. 

Zeitschr.  fiir  Hygiene,  Bd.  X.,  1891,  p.  267. 
Heilversuche  an  Tetanuskranken  Thieren.     Zeitschr.  fflrHyg., 

XII.,  256,  1893. 
Klipstein,  E. :  Ueber  die  Wirkung  giftfreierTetanuskulturen.     H3'gien. 

Rundschau,  1898,  p.  1. 
Renou  :  Deux  cas  de  tetanos  traites  par  des  injections  de  sang  antitox- 

ique.     Ann.  de  I'Inst.  Pasteur,  1893,  p.  383. 
Schiitz  :  Versuche  zur  Immunisirung  von  Pferden  und  Schafen  gegen 

Tetanus.     Zeitschr.  fiir  Hyg.,  XII.,  58,  1893. 
Tizzoni  e  Cattani :  Ueber  das  Tetanusgift.     Centralbl.  ftlr  Bakteriol. , 

Bd.  VIII. ,  1890,  p.  69. 
Ueber  die  Art  einen  Thiere  die  Iramunitat  gegen  Tetanus  zu 

tlbertragen.     Centralbl.  fiir  Bakteriol.,  Bd.  IX.,  1891,  p.  189. 
Ueber   die  Eigenschaften   des   Tetanus-Antitoxins.      Centralbl. 

ftlr  Bakteriol.,  Bd.  IX.,  1891,  p.  685. 
Fernere  Untersuchungen  tiber  das  Antitoxin  des  Tetanus.     Cen- 
tralbl. fUr  Bakteriol.,  Bd.  X.,  1891,  p.  33. 
Alcune  questioni  relative  all'   immunlta  pel   tetano.     Riforma 

Med.,  1893,  No.  193,.  193. 
Sulla  transmissione  ereditaria  dell'  immunita  contro  il  tetano. 

Riforma  Med.,  1893,  No.  94. 

Suir  importanza  della  milza  nell'  immunizzazione  sperimentale 

del  coniglia  contro  il  tetano.     Riforma  Med.,  1893,  No.  49. 

Esperienze  sulla  vaccinazioue  del  cavallo  contro  11  tetano.     La 

Rif.  Med.,  1893,  No.  131. 


270  IMMUNITY   AND   SERU3I-TIIERAPY. 

Tizzoni  e  Cattani  :  Weitere  Experiraentelle  Untersucliungen  tiber  die 
Imniunitat  gegeu  Tetanus.  Berl.  klin.  AVocbenschr.,  1893,  p.  1185, 
1215, 1245,  1265. 

Neue  Unteisucbungen  ul)er  die  Vaccination  des  Pferdes  gegen 

Tetanus.     Berl.  klin.  Wcbscbr.,  1894,  No.  32. 

Nuove  esperienze   sulla  vaccinazione  del  cavallo  contro  il  te- 

tano.     Gazz.  d.  Ospit.,  1894,  p.  505. 

Weitere  Experimentelle   Untersucbungen  tiber  die  Inimunitiit 

gegen  Tetanus.     Berl.  klin.  Wcliscbr.,  1894,  p.  64. 

Trevisan,  A.  :  Sulla  inalterata  virulenza  del  materiale  tetanigeno  con- 
servato  in  glicerina.     Rev.  Veneta  di  Sci.  Med.,  1892,  p.  129. 

Vaillard  :  Sur  rimmuuite  contre  le  tetanos.  Corapte-rendu,  Sec.  de 
Biol.,  1891,  No.  7. 

Sur  les  proprietes  du  serum  des  aniraaux  refractaires  an  teta- 
nos.    La  Semaine  Med.,  1891,  p.  254. 

Sur  rinoculation  aux  animaux  du  bacille  tetanique  depourvu  de 

toxine.     Le  Bull.  Med.,  1891,  p.  901. 

Sur  quelques  points  concernant  rimmunite  contre  le  tetanos. 

Ann.  de  I'lnst.  Pasteur,  1892,  p.  676. 

De  Paction  des  bumeurs  d'un  animal  immunise  contre  le  tetanos 


sur  le  virus  de  cette  maladie.     Ann.  de  I'lnst.  Pasteur,  1892,  p.  676. 
Wernicke  :  Ueber  Bebring's  Blutserumtherapie  bei  Tetanus.     Abst.  in 

Centralbl.  Mr  Bakteriol.,  XV.,  898,  1894. 
Wladimiroflf,  A.  :  Ueber  die  Antitoxinerzeugende  und  Immunisirende 

Wirkung  des  Tetanusgiftes  bei  Tbieren.     Zeitscbr.  ftLr  Hyg.,  XV., 

405,  1893. 


XX. 

TUBERCULOSIS. 

The  announcement  of  the  discovery  of  the  tubercle 
bacillus  was  made  by  Koch,  in  March,  1882,  at  a  meeting" 
of  the  Physiological  Society  of  Berlin.  At  the  same 
time  satisfactory  experimental  evidence  was  presented  as 
to  its  etiological  relation  to  tuberculosis  in  man  and  in 
the  susceptible  lower  animals,  and  its  principal  biologi- 
cal characters  were  given. 

This  achievement,  the  result  of  patient  and  intelligent 
scientific  investigation,  will  always  rank  as  one  of  the 
most  important  in  the  history  of  medicine.  The  previ- 
ous demonstration  by  Villemin  (1865)  —  confirmed  by 
Cohnheim  (1877)  and  others — that  tuberculosis  might  be 
induced  in  healthy  animals  by  inoculations  of  tubercu- 
lous material,  had  paved  the  way  for  this  great  discov- 
ery, and  advanced  pathologists  were  quite  prepared  to 
accept  it.  The  more  conservative  have  since  been 
obliged  to  yield  to  the  experimental  evidence,  which  has 
received  confirmation  in  all  parts  of  the  world.  To-day 
it  is  generally  recognized  that  tuberculosis  is  a  specific 
infectious  disease  due  to  the  tubercle  bacillus. 

As  evidence  of  the  thorough  nature  of  Koch's  personal 
researches  in  advance  of  his  first  public  announcement, 
we  give  the  following  resume  of  his  investigations  : 

In  nineteen  cases  of  miliary  tuberculosis  the  bacilli 


272  IMMUNITY    AND    SERUM-TIIEKAPY. 

were  found  in  the  tubercular  nodules  in  every  instance  ; 
also  in  twenty-nine  cases  of  pulmonary  phthisis,  in  the 
sputum,  in  fresh  cheesy  masses,  and  in  the  interior  of 
recently  formed  cavities  ;  in  tuberculous  ulcers  of  the 
tongue,  tuberculosis  of  the  uterus,  testicles,  etc.  ;  in 
twenty-one  cases  of  tuberculous — scrofulous — lymi^hatic 
glands ;  in  thirteen  cases  of  tuberculous  joints  ;  in  ten 
cases  of  tubercular  bone  affections  ;  in  four  cases  of 
lupus  ;  in  seventeen  cases  of  jyerhucht  in  cattle.  His  ex- 
perimental inoculations  were  made  upon  two  hundred 
and  seventy-three  guinea-pigs,  one  hundred  and  live  rab- 
bits, forty-four  field-mice,  twenty-eight  white  mice,  nine- 
teen rats,  thirteen  cats,  and  upon  dogs,  pigeons,  chick- 
ens, etc.  Very  extensive  comparative  researches  were 
also  made,  which  convinced  him  that  the  bacillus  which 
he  had  been  able  to  demonstrate  in  tuberculous  sputum 
and  tissues  by  a  special  mode  of  staining  was  not  to  be 
found  in  the  sputa  of  healthy  persons,  or  of  those  suffer- 
ing from  non-tubercular  pulmonary  affections,  or  in  or- 
gans and  tissues  involved  in  morbid  processes  of  a  differ- 
ent nature. 

The  tubercle  bacillus  is  a  stncf  j)arasite,  and  its  bio- 
logical characters  are  such  that  it  could  scarcely  find 
natural  conditions,  outside  of  the  bodies  of  living  ani- 
mals, favorable  for  its  multiplication.  It  therefore  does 
not  grow  as  a  saprophyte  under  ordinary  circumstances. 
But  it  has  been  noted  by  Eoux  and  Nocard  that  when 
it  has  been  cultivated  for  a  time  in  artificial  media  con- 
taining glycerin  it  may  grow  in  a  plain  bouillon  of  veal 
or  chicken,  in  which  media  it  fails  to  develop  when  in- 
troduced directly  from  a  culture  originating  from  the 
body  of  an  infected  animal.  This  would  indicate  the 
possibility  of  its  acquiring  the  ability  to  grow  as  a  sapro- 


TUBERCULOSIS.  273 

pliyte  ;  and  we  can  scarcely  doubt  that  at  some  time  in 
the  past  it  was  a  true  saprophyte.  The  experiments  of 
Nuttall  indicate  that  the  bacillus  may  multiply,  under 
favorable  temperature  conditions,  in  tuberculous  sputum 
outside  of  the  body.  And  it  is  extremely  probable  that 
multiplication  occurs  in  the  muco-purulent  secretion 
which  accumulates  in  pulmonary  cavities  in  phthisical 
patients.  In  these  cavities  its  development  may,  in  a 
certain  sense,  be  reg-arded  as  saprophytic,  as  it  feeds 
upon  non-living*  org-anic  material. 

Metschnikoff  states  that  when  kept  at  a  temperature 
of  42°  C.  for  some  time  the  tubercle  bacillus  underg-oes  a 
notable  diminution  in  its  i3athog"enic  power,  and  that 
when  kept  at  a  temperature  of  43°  to  44°  C.  it  after  a 
time  only  induces  a  local  abscess  when  injected  subcu- 
taneously  into  gruinea-pigs.  The  experiments  of  Lote 
also  indicate  that  an  "  attenuation  of  virulence  "  has  oc- 
curred in  the  cultures  preserved  in  Koch's  laboratory, 
orig'inating"  in  1882  from  the  lungs  of  a  tuberculous  ape. 
The  author  named  made  experiments  with  cultures  from 
this  source  (ninetieth  to  ninety-fifth  successive  culture), 
and  at  the  same  time  with  a  culture  obtained  from  Koux, 
of  Pasteur's  laboratory.  Rabbits  inoculated  with  cultures 
from  the  last-mentioned  source  developed  a  hectic  fever 
at  the  end  of  two  weeks,  and  died  tuberculous  at  the  end 
of  twenty-one  to  thirty-nine  days.  Twelve  rabbits  were 
inoculated  with  the  cultures  from  Koch's  laboratory  ;  the 
injections  were  made  either  subcutaneously,  into  a  vein, 
into  the  pleural  cavity,  or  into  the  cavity  of  the  abdo- 
men. No  elevation  of  temperature  occurred  in  any  of 
the  animals,  and  they  were  found  at  the  end  of  a  month 
to  have  increased  in  weig-ht.  At  the  end  of  six  weeks 
one  of  them  was  killed  and  tubercular  nodules  were 
18 


274  IMMUNITY   AND   SERUM-THERAPY. 

found  in  various  organs.  The  remaining  animals  were 
killed  at  the  end  of  one  hundred  and  forty-four  to  one 
hundred  and  forty-eight  days.  The  two  inoculated  sub- 
cutaneouslj^  presented  no  sign  of  general  tuberculosis, 
but  a  small  yellow  nodule  containing  bacilli  was  found 
at  the  point  of  inoculation.  Those  inoculated  by  injec- 
tion into  a  vein  showed  one  or  two  nodules  in  the  lungs 
containing  a  few  bacilli.  In  Koch's  original  experiments 
rabbits  were  killed  by  intravenous  inoculation  of  his  cult- 
ures in  from  thirteen  to  thirty-one  days.  That  this  at- 
tenuation of  virulence  depends  upon  a  diminished  pro- 
duction of  a  toxic  product  to  which  the  bacillus  owes  its 
pathogenic  power  appears  to  be  very  certain,  in  view  of 
the  fact  that  the  late  cultures  in  a  series  have  a  more 
vigorous  and  abundant  development  than  the  more  patho- 
genic cultures  obtained  directly  from  the  animal  body. 

The  discovery  by  Koch  of  a  toxin  in  cultures  of  this  ba- 
cillus, which  is  soluble  in  glycerin,  and  which  in  very 
minute  doses  produces  febrile  reaction  and  other  decided 
symptoms  when  injected  subcutaneously  into  tubercu- 
lous animals,  must  rank  as  one  of  the  first  importance  in 
scientific  medicine,  whatever  the  final  verdict  may  be  as 
to  its  therapeutic  value  in  tubercular  diseases  in  man. 

The  toxic  substance  contained  in  Koch's  glycerin  ex- 
tract from  cultures  of  the  tubercle  bacillus,  now  gener- 
ally known  under  the  name  of  tuberculin,  is  sohible  in 
water,  insoluble  in  alcohol,  and  passes  readily  through 
dialyzing  membranes.  It  is  not  destroyed  by  the  boil- 
ing temperature.  According  to  the  chemical  examina- 
tion of  JoUes,  the  "  lymph  "  contains  fifty  per  cent,  of 
water  and  does  not  contain  alkaloids  or  cyanogen  com- 
pounds. It  contains  albuminates,  which  are  thrown 
down  as  a  voluminous  white  precipitate  by  tannic  acid, 


TUBERCULOSIS.  275 

and  are  redissolved  by  hot  water  containing  sodium 
chloride  and  very  diluted  potash  solution.  The  elemen- 
tary analysis  gave  N  5.90  per  cent.,  C  35.19  per  cent.,  and 
H  7.02  per  cent.  The  results  obtained  are  believed  to 
show  that  the  active  substance  present  in  the  lymph  is  a 
toxalbumin.  In  experiments  made  with  Koch's  lymph 
in  Pasteur's  laboratory  by  Bardach,  a  very  decided  ele- 
vation of  temperature  was  produced  in  tuberculous 
guinea-pigs  by  the  subcutaneous  injection  of  0.1  gm.,  and 
a  fatal  result  by  the  injection  of  0.2  to  0.5  gm.  In  man  a 
decided  febrile  reaction  is  produced  in  tuberculous  pa- 
tients by  very  much  smaller  doses — 0.001  c.c. 

Hammerschlag,  in  his  chemical  researches,  found  that 
the  tubercle  bacillus  yields  a  larger  proportion  of  sub- 
stances soluble  in  alcohol  and  ether  than  any  other  ba- 
cilli tested  (twenty-seven  per  cent.).  The  alcoholic  ex- 
tract contains  fat,  lecithin,  and  a  toxic  substance  which 
produces  convulsions  in  rabbits  and  guinea-pigs.  The 
portion  insoluble  in  alcohol  and  ether  contains  cellulose 
and  an  albuminoid  substance.  No  ptomaines  were  found, 
but  a  toxalbumin  was  isolated,  which  caused  an  elevation 
of  temperature  in  rabbits  of  1°  to  2°  C,  lasting  for  a  day 
or  two. 

Koch  (1891)  has  given  a  full  account  of  his  method  of 
preparing  crude  tuberculin,  and  also  the  process  by 
which  he  obtains  from  this  a  tuberculin  which  appears  to 
be  pure,  or  nearly  so.  To  obtain  considerable  quantities 
of  the  crude  product  the  tubercle  bacillus  is  cultivated  in 
an  infusion  of  calves'  flesh,  or  of  beef  extract  to  which 
one  per  cent,  of  peptone  and  four  to  live  per  cent,  of 
glycerin  have  been  added.  This  culture  liquid  must  be 
made  slightly  alkaline,  and  it  is  placed  in  flasks  with  a 
flat  bottom,  which  should  not  be  more  than  half  filled— 


27G  IMMUNITY   AND   SERUM-TIIEIIAPY. 

30  to  50  c.c.  The  inoculation  is  made  upon  the  sur- 
face with  small  masses  from  a  culture  upon  blood-serum 
or  g-lycerin  agar.  By  accident  Koch  discovered  that 
these  masses  fioating  upon  the  surface  give  rise  to  an 
abundant  development,  and  to  the  formation  of  a  toler- 
ably thick  and  dry  white  layer,  which  finally  covers  the 
entire  surface.  At  the  end  of  six  to  eight  weeks  develop- 
ment ceases,  and  the  layer  after  a  time  sinks  to  the  bot- 
tom, breaking  up  meanwhile  into  fragments.  These 
cultures,  after  their  purity  has  been  tested  by  a  micro- 
scopical examination,  are  poured  into  a  suitable  vessel 
and  evaporated  to  one-tenth  the  original  volume  over  a 
water-bath.  The  liquid  is  then  filtered  through  porce- 
lain. The  crude  tuberculin  obtained  by  this  process  con- 
tains from  forty  to  fifty  per  cent,  of  glycerin,  and  conse- 
quently is  not  a  suitable  medium  for  the  develojDment  of 
saprophytic  bacteria,  if  they  should  by  accident  be  intro- 
duced into  it.  It  keeps  well  and  preserves  its  activity 
indefinitely. 

From  this  crude  tuberculin  Koch  has  obtained  a  white 
precipitate,  with  sixty  per  cent,  alcohol,  which  has  the 
active  properties  of  the  crude  tuberculin  as  originally 
prepared.  This  is  fatal  to  tuberculous  guinea-pigs  in 
doses  of  2  to  10  milligm.  It  is  soluble  in  water  and  in 
glycerin,  and  has  the  chemical  reactions  of  an  albumi- 
nous body.  In  preparing  it  one  volume  and  a  half  of 
absolute  alcohol  is  added  to  one  volume  of  the  crude 
tuberculin,  and,  after  stirring  it  to  secure  uniform  admixt- 
ure, this  is  put  aside  for  twenty -four  hours.  At  the  end 
of  this  time  a  flocculent  deposit  will  be  seen  at  the  bot- 
tom of  the  vessel.  The  fluid  above  this  is  carefully 
poured  off ;  and  an  equal  quantity  of  sixty  per  cent,  alco- 
hol is  poured  into  the  vessel  for  the  purpose  of  washing 


TUBEKCULOSIS.  277 

the  precipitate.  This  is  again  allowed  to  settle,  and  the 
procedure  is  repeated  three  or  four  times,  after  which  the 
I)recipitate  is  washed  with  absolute  alcohol.  It  is  then 
placed  upon  a  filter  and  dried  in  a  vacuum  exsiccator. 

The  "  tuberculocidin  "  of  Klebs  is  a  purified  tuberculin 
obtained  by  precipitation  with  alcohol.  The  precipitate 
is  washed  in  chloroform  and  then  dissolved  in  a  mixture 
of  carbolic  acid  and  glycerin. 

Bujwid  (1894)  prepares  tuberculin  as  follows  :  He  uses 
cultures  on  glycerin  agar  or  in  glycerin  bouillon  which 
have  been  kept  at  a  suitable  temperature  for  five  to  eight 
weeks.  The  glycerin-agar  cultures  are  treated  with  dis- 
tilled water  by  which  the  tuberculin  is  extracted.  After 
adding  the  water  the  test-tubes  are  kept  in  a  cool  place 
for  twenty-four  hours,  and  this  is  repeated  two  or  three 
times.  The  extract  from  the  agar  cultures  or  the  bouil- 
lon cultures  is  then  sterilized  by  exposure  for  from  five 
to  ten  minutes  to  a  temj)erature  of  100°  C  ;  then  filtered 
through  a  Chamberland  filter  ;  then  evaporated  at  a  low 
temperature  to  a  sirup -like  consistence.  When  this 
crude  tuberculin  is  dropped  into  ten  times  its  volume  of 
strong  alcohol  a  brown  precipitate  is  thrown  down  which 
contains  the  active  principle.  From  the  tubercle  bacilli 
obtained  by  filtering  his  cultures  Bujwid  also  obtained  an 
active  substance  which  in  the  dose  of  2  milligm.  caused  an 
elevation  of  2°  C.  in  the  temperature  of  an  infected  guinea- 
pig.  This  substance  was  obtained  by  digesting  the  ba- 
cilli for  two  months  in  glycerin  and  water  (three  per 
cent,  of  glycerin),  filtering  and  evaporating  the  extract, 
and  precipitation  in  six  volumes  of  ninety-five  per  cent, 
alcohol.  The  precipitate  when  dried  was  in  the  form  of 
a  white  powder. 

Helman  (1894)  obtains  tuberculin  from  potato  cultures. 


278  IMMUNITY   AND    SERUM-TIIEIIAP V. 

The  sections  of  potato  are  neutralized  by  leaving-  them 
for  half  an  hour  in  a  solution  of  one-half  to  one  per 
cent,  of  bicarbonate  of  soda,  after  which  they  are  steril- 
ized for  twenty  minutes  in  the  autoclave  at  120°  C.  The 
best  results  were  obtained  when  the  potatoes  were  wet 
with  a  five  per  cent,  solution  of  g-lycerin.  The  sections 
of  potato  were  iDlaced  in  Petri's  dishes  upon  blotting- 
paper  wet  with  a  sublimate  solution,  and  the  dishes  con- 
taining* the  cultures  were  surrounded  with  cotton  wet 
with  the  same  solution.  The  cultures  w^ere  subsequently 
treated  with  distilled  water,  to  extract  the  active  princi- 
ple, which  was  also  obtained  from  the  bacilli  bj^  mixing- 
them  with  glycerin  in  the  proportion  of  1  to  10. 

Numerous  experiments  have  been  made  with  dead  tu- 
bercle bacilli,  as  well  as  with  the  toxic  products  devel- 
oped in  cultures.  Hericourt  and  Richet  (1890)  found  by 
experiment  that  old  cultures  heated  to  80°  C,  several  days 
in  succession,  when  injected  into  a  vein  in  rabbits,  in  the 
dose  of  10  to  20  c.c,  caused  the  death  of  these  animals. 
Smaller  doses  from  which  the  animals  recovered  seemed 
to  make  them  less  susceptible  to  infection  than  control 
animals,  but  the  number  of  experiments  was  too  limited 
to  establish  this  as  a  fact.  In  a  subsequent  (1891)  com- 
munication the  authors  named  claim  to  have  succeeded 
in  immunizing  rabbits  by  injecting  filtered  and  sterilized 
cultures  of  the  tubercle  bacillus,  either  subcutaneously  (5 
to  15  c.c.)  or  into  a  vein  (20  to  40  drops).  The  injections 
were  repeated  every  second  or  third  day  for  a  period  of 
fifteen  days,  after  which  the  test  inoculation  was  made 
with  a  culture,  obtained  from  a  tuberculous  cow  in  one 
series,  and  from  tuberculous  fowls  in  another.  Four  vac- 
cinated rabbits  in  the  first  series  escaped  general  tubercu- 
losis, while  four  out  of  eight  control  animals  died  tuber- 


TUBERCULOSIS.  279 

culous.  In  the  second  series  five  vaccinated  animals 
resisted  infection  and  three  out  of  four  control  animals 
died  tuberculous. 

De  Schweinitz  has  recently  (1894)  reported  the  results 
of  experiments  with  attenuated  cultures  of  the  tubercle 
bacillus,  and  has,  apparently,  succeeded  in  conferring  im 
inunity  upon  guinea-pigs  by  inoculations  with  such  cult- 
ures. 

Klebs  (1891),  in  experiments  on  guinea-pigs  and  rab- 
bits, convinced  himself  that  the  fatal  result  of  an  inocula- 
tion with  tubercle  bacilli  (in  the  cavity  of  the  abdomen 
or  subcutaneously  in  guinea  -  pigs,  and  in  the  eye  in 
rabbits)  was  greatly  delayed  by  injections  of  Koch's 
tuberculin  (0.3  to  0.5  c.c.)  either  before  or  after  infec- 
tion. 

Baumgarten  (1891),  in  experiments  upon  rabbits  inocu- 
lated with  tubercle  bacilli  in  the  anterior  chamber  of  the 
eye,  failed  to  obtain  favorable  results  from  treatment  with 
Koch's  tuberculin  given  in  considerable  doses  (0.5  to  1 
gm.)  either  before  or  after  infection. 

The  results  reported  in  the  same  year  by  Gramatschi- 
koff,  b}^  Popoff,  by  Alexander  and  by  Gasparini  and  Mer- 
canti,  were  also  unfavorable  as  regards  an  immunizing  or 
curative  effect  from  inoculations  of  tuberculin  in  rabbits. 
Diinitz,  on  the  contrary,  arrives  at  the  conclusion  that 
when  early  treatment  is  instituted  iris  tuberculosis  may 
be  arrested  and  cured,  and  the  more  recent  experiments 
of  Trudeau  (1893)  give  support  to  this  conclusion.  Baum- 
garten, however,  insists  that  the  tuberculin  treatment  does 
not  prevent  metastasis  to  the  lungs  after  inoculations  in 
the  anterior  chamber  of  the  eye. 

Pfuhl  (1891)  treated  forty-seven  infected  guinea-pigs 
and  at  the  date  of  his  report  forty -four  had  died  tuber- 


280  IMMUNITY    AND   SERUM-TIIEKAPY. 

culoiis,  but  the  date  of  death  was  somewhat  postponed 
by  the  treatment.  The  auimals  not  treated  succumbed  at 
the  end  of  eight  weeks  (average  of  all  controls),  and  those 
treated  with  small  doses  of  tuberculin  lived,  on  the 
average,  ten  weeks.  With  larger  doses  still  more  favor- 
able results  were  obtained — four  lived  on  an  average 
twelve  weeks,  and  three  were  still  living,  eleven,  fifteen, 
and  sixteen  weeks  after  infection,  at  the  date  of  jjubli- 
cation. 

Kitasato  (1892)  also  obtained  favorable  results  in  the 
treatment  of  infected  guinea-pigs,  and  arrives  at  the  con- 
clusion that  guinea-pigs  Avhich  have  been  cured  by  the 
treatment  are  not  susceptible  to  a  second  infection,  for  a 
certain  time  at  least. 

Bujwid  (1892),  in  experiments  upon  guinea-pigs,  found 
that  infected  animals  which  received  from  0.05  to  0.1  gm. 
of  tuberculin  within  three  hours  showed  an  elevation  of 
temperature  of  1.5°  to  2°  C.  Thirteen  infected  guinea- 
pigs  treated  with  tuberculin  lived  from  two  and  a  half  to 
eight  months,  while  all  of  the  control  animals  (eighteen) 
died  in  from  six  to  nine  weeks.  The  animal  which  sur 
vived  eight  months  was  found  not  to  be  tuberculous  but 
presented  evidence  of  recovery  from  a  former  tuberculous 
process.  In  two  rabbits  inoculated  in  the  anterior  cham- 
ber the  iris  tuberculosis  was  favorably  influenced  by  the 
tuberculin  treatment,  but  general  infection  occurred  and 
the  animals  died  about  the  same  time  as  the  controls. 
Three  apes  were  treated  without  any  apparent  result ; 
they  all  died  within  two  months  after  infection. 

The  experiments  of  Gramatschikoff,  Czaplewski  and 
Roloff,  and  of  Yamagiva,  published  in  1892,  show  that  the 
tuberculin  treatment  does  not  cure  tuberculous  infection 
in  inoculated    guinea-pigs    and    rabbits,  and    that    the 


TUBERCULOSIS.  281 

bacilli  retain  their  vitality  in  such  animals  in  spite  of  the 
most  persistent  treatment. 

Hericonrt  and  Ricliet  (1892),  in  experiments  made  for 
the  purpose  of  immunizing  animals  against  tuberculous 
infection,  failed  to  obtain  positive  results  in  the  most 
susceptible  species — guinea- jiigs,  rabbits,  and  apes — but 
claim  to  have  succeeded  in  immunizing  dogs  by  intra- 
venous injections  of  cultures  of  the  bacillus  of  tubercu- 
losis in  fowls.  Animals  which  had  been  so  treated  after 
an  interval  of  two  to  six  mouths  received  an  intravenous 
injection  of  1  c.c.  of  a  culture  of  the  bacillus  tuberculosis 
from  man.  This  was  fatal  to  "  non-vaccinated  "  dogs,  as  a 
rule,  in  about  three  weeks,  but  the  "  vaccinated  "  animals 
survived  the  injection. 

The  results  obtained  by  Trudeau  (1893)  are  of  such  in- 
terest that  we  shall  quote  in  exteiiso  what  he  says  with 
reference  to  x)reventive  inoculations  : 

"  Antitubercular  inoculation  was  first  tried  by  Falk  in 
1883,  and  all  attempts  in  this  direction  have  resulted  un- 
til recently  in  but  an  unbroken  record  of  failures.  In 
1890  I  added  my  name  to  the  list  of  those  who  found 
it  impossible  to  produce  immunity  in  animals  by  this 
method.  In  1890,  Martin  and  Grancher,  and  Courmont 
and  Dor,  claimed  to  have  iiroduced  in  rabbits  a  certain 
degree  of  immunit}^  by  previous  inoculation,  after  Pas- 
teur's hydrophobia  method,  of  avian  tubercle  bacilli  of 
graded  and  increasing  virulence.  These  vaccinations 
were,  however,  frequently  fatal  to  the  animals,  and  the 
immunity  obtained  was  but  slight.  Richet  and  Hericonrt 
have  since  claimed  to  produce  complete  immunity  in 
dogs  by  intravenous  inoculations  of  bird  tubercle  bacilli. 
These  experimenters  found  that  though  harmless  to  the 
dog  when  first  derived  from  the  chicken,  bird  bacilli,  by 
long  cultivation  in  liquid  media,  become  pathogenic  for 
this  animal,  and  by  thus  grading  the  virulence  of  the  in- 


282  IMMUNITY    AND   SERUM-THERAPY. 

jections  complete  immunity  against  any  form  of  tubercu- 
lar infection  Avas  produced  in  tlie  dog.  As  yet  these  strik- 
ing results  have  not  been  continued.  The  animals  which 
I  now  present  to  you  illustrate  an  attempt  I  have  made 
along  the  same  line  to  produce  immunity  in  the  rabbit. 
Cultures  grown  directly  from  the  chicken's  lesions  in 
bouillon  for,  first,  five  weeks,  then  six  months,  were  twice 
injected  subcutaneously  at  intervals  of  twenty-one  days 
in  doses  of  0.025  and  0.05,  and  a  third  injection  of  a  still 
older  culture  was  occasionally  given.  About  one  in  four 
of  the  rabbits  died  within  three  months,  profoundly  ema- 
ciated, but  without  any  visible  tubercular  lesions.  The 
remaining  animals  recovered  and  were  apparently  in 
good  health,  when,  together  with  an  equal  number  of  con- 
trols, they  were  inoculated  in  the  anterior  chamber. of  the 
eye  with  cultures  of  Koch's  bacillus  derived  from  the 
tuberculous  lesions  of  the  rabbit,  and  cultivated  about 
three  months  on  glycerin  agar.  The  results  of  these  in- 
oculations ijresent  many  points  of  interest.  In  the  con- 
trols, as  is  usually  the  case,  if  the  operation  has  been 
done  carefully  and  aseptically,  and  with  a  moderate 
amount  of  dilute  virus,  two  days  after  the  introduction  of 
the  virulent  material  in  the  eye  little  or  no  irritation  is 
observed,  and  little  is  to  be  noticed  for  two  weeks,  when 
a  steadil}^  increasing  vascularity  manifests  itself,  small 
tubercles  appear  on  the  iris,  which  gradually  coalesce  and 
become  cheesy,  intense  iritis  and  general  inflammation  of 
the  structures  of  the  eye  develop,  the  inoculation  wound 
becomes  cheesy,  and  in  six  to  eight  weeks  the  eye  is  more 
or  less  com^iletely  destroyed  and  the  inflammation  begins 
to  subside.  The  disease,  however,  remains  generally 
localized  in  the  eye  for  many  months,  and  even  perma- 
nently. In  the  vaccinated  animals,  on  the  contrary,  the 
introduction  of  the  virulent  bacilli  at  once  gives  rise  to  a 
marked  degree  of  irritation.  On  the  second  day  the 
vessels  of  the  conjunctiva  are  tortuous  and  enlarged, 
whitish  specks  of  fibrinous-looking  exudation  appear  in 
the  iris  and  in  the  anterior  chamber,  and  more  or  less  in- 


TUBERCULOSIS.  283 

tense  iritis  supervenes,  but  at  the  end  of  the  second  to 
the  third  week,  when  the  eyes  of  the  controls  begin  to 
sliow  progressive  and  steadily  increasing-  evidence  of 
iufiammatory  reaction,  the  irritation  in  those  of  the 
vaccinated  animals  beg-ins  slowly  to  subside  and  the  eyes 
to  mend.  The  vascularity^  is  less,  the  whitish  spots  of 
fibrinous  material  appear  smaller,  the  structures  of  the 
eye  become  clearer,  the  inoculation  wound  is  but  a  bluish 
fibrous  scar,  until  in  from  six  to  twelve  weeks,  in  success- 
ful cases,  all  irritation  has  disappeared  and  the  eyes 
present,  as  in  the  animals  I  now  show  you,  but  fibrous 
evidence  of  the  traumatism  and  the  inflammatory  pro- 
cesses which  have  been  set  up  by  the  inoculation.  In  all 
the  controls,  as  you  see,  the  inoculation  wound  is  cheesy 
and  the  cornea  and  iris  are  more  or  less  destroyed  by 
tubercle  and  cheesy  areas. 

"  Some  of  the  protected  animals  slowly  relapse,  and  the 
one  I  now  show  you  has  small  tubercles  g-rowing  on  the 
iris ;  but  even  in  such  eyes  the  entire  absence  of  casea- 
tion is  noticeable,  and  the  disease  progresses  almost  im- 
perceptibly. I  have  repeated  this  experiment  on  three 
sets  of  rabbits  with  about  the  same  results  each  time. 
The  vaccinations  as  practised  are  of  themselves,  in  some 
instances,  fatal,  but  the  fact  remains  that  where  recovery 
takes  place  a  marked  degree  of  immunity  has  been  ac- 
quired. I  do  not  lay  any  claim,  therefore,  to  have  pro- 
duced a  complete  or  permanent  immunity  by  a  safe 
method,  but  it  seems  to  me  that  these  eyes  constitute  a 
scientific  demonstration  of  the  fact  that  in  rabbits  pre- 
ventive inoculation  of  bird-tubercle  bacilli  can  retard, 
and  even  abort,  an  otherwise  progressive  localized  tuber- 
cular process  so  completely  as  to  prevent  destruction  of 
the  tissues  threatened,  and  that  the  future  study  of  anti- 
tubercular  inoculation  may  not  be  as  entirely  hopeless  as 
it  has  until  recently  appeared." 

We  cannot  attempt  to  review  the  extensive  literature 
relatiuo-  to    the   treatment  of    tuberculosis  in  man  by 


284  IM3IUNITY   AND   SERUM-THERAPY. 

means  of  Koch's  tuberculin  and  the  various  preparations 
obtained  from  cultures  of  the  tubercle  bacillus.  We  may, 
however,  briefly  summarize  the  results  reported  by  the 
statement  that  the  clinical  evidence  shows  that  in  early 
and  properl3^  selected  cases  the  treatment  may  exercise  a 
favorable  influence  on  the  progress  of  pulmonary  tuber- 
culosis ;  but  that  it  can  by  no  means  be  accepted  as  a 
specific  for  this  disease,  and  in  advanced  cases  its  inju- 
dicious use  may  do  much  harm. 

It  is  evident  that  in  a  disease  in  which  recovery  some- 
times occurs  independently  of  treatment,  and  in  clinical 
experiments  upon  patients  who  differ  in  their  suscepti- 
bility to  tubercular  infection  and  in  other  important  par- 
ticulars, and  in  the  absence  of  "  controls  "  such  as  we  find 
it  necessary  to  employ  in  experimenting-  upon  the  lower 
animals,  the  reported  results  of  any  plan  of  treatment 
must  be  accepted  with  great  caution. 

Having  this  in  view  we  may  refer  to  the  very  favorable 
report  of  Schiess-Bey  and  Kartulis  (1893).  Out  of  foi-tv- 
eight  cases  treated  during  a  period  of  two  years,  sixteen 
are  said  to  have  been  permanently  cured.  The  authors 
referred  to  conclude  from  their  experience  that  commenc- 
ing pulmonary  tuberculosis  may  be  surely  cured  by  the 
use  of  tuberculin  in  the  course  of  three  to  four  months ; 
also  that  more  advanced  cases  may  be  cured  if  the  treat- 
ment is  persisted  in  for  six  months  to  a  year.  Even  cases 
having  small  cavities  may,  under  exceptionally  favorable 
conditions,  be  cured.  But  when  large  cavities  exist,  with 
hectic  fever  and  night-sweats,  the  treatment  is  of  no  avail. 

The  authors  referred  to  consider  the  treatment  as  de- 
void of  danger  whf^n  very  small  doses  are  employed  at 
the  outset,  but  these  are  not  sufficient  to  effect  a  cure 
and  must    be   gradually    increased.      Usually    yV   of    a 


TUBERCULOSIS.  285 

milligr.  was  given  at  lirst,  but  sometimes  1  milligi". 
was  g-iven.  These  doses  did  not  produce  a  decided  reac- 
tion, and  it  was  the  aim  of  the  authors  to  avoid  such  reac- 
tion by  giving  comparatively  small  doses  at  the  outset 
and  carefully  increasing  them.  Finally,  doses  of  100  mil- 
ligr.  were  reached,  after  which  the  dose  was  again  grad- 
ually diminished  to  10  milligr.,  and  subsequently  in- 
creased again  to  the  maximum.  Of  the  48  cases  treated 
33  were  pulmonary  and  13  "  surgical "  (tuberculosis  of 
bones  and  skin).  In  the  last-mentioned  group  of  cases 
the  results  led  the  authors  to  formulate  the  following 
conclusions  :  "  Certain  forms  of  bone  and  joint  tuberculo- 
sis, as  well  as  gland  tuberculosis,  are  cured  more  quickly 
by  the  use  of  tuberculin  in  combination  with  surgical 
measures  than  by  surgical  measures  alone."  Another 
conclusion  which  may  indicate  an  important  element  in 
the  success  ascribed  to  the  tuberculin  treatment  is  that 
"  the  Egyptian  climate  is  especially  favorable  for  the 
tuberculin  treatment." 

Escherich  (1892)  reports  that  in  his  experience  pulmo- 
nary tuberculosis  was  rather  unfavorably  influenced  by 
the  tuberculin  treatment  than  otherwise,  and  the  results 
obtained  in  the  treatment  of  tuberculous  joints  and 
glands  could  scarcely  be  considered  favorable.  But  in 
the  various  forms  of  skin  tuberculosis  a  curative  effect 
was  manifested. 

Klebs  (1892)  reports  that  out  of  33  cases  treated  by 
himself  with  tuberculocidin  (Klebs' s)  8  were  cured  and  21 
improved.  In  75  cases  treated  by  his  colleagues  6  were 
cured  and  24  improved.  He  therefore  concludes  that 
this  preparation  is  a  very  effective  therapeutic  agent  for 
the  treatment  of  tuberculosis. 

Kaatzer  (1891)  reports  44  cases  of  pulmonary  tubercu- 


286  IMMUNITY    AND   SERUM-THERAPY. 

losis  treated.  Of  these  14  died,  16  were  discliarg-ed 
cured  (?),  9  improved  under  treatment,  4  did  not  improve, 
and  1  remained  under  treatment  at  the  date  of  publica- 
tion. The  commeneing-  dose  was  from  j\  to  1  milligr.  ; 
the  duration  of  treatment  was  from  six  to  fifty  -  two 
weeks  ;  the  average  quantity  of  tuberculin  administered 
to  each  patient  1,535  millig-r. 

In  the  last  published  report  of  the  Adirondack  Cottage 
Sanitarium  (1894)  Trudeau  says  : 

"  The  medical  report  continues  to  indicate  that  for  a  cer- 
tain class  of  cases  the  best  and  most  permanent  results  are 
obtained  by  the  addition  of  treatment  by  modified  tubercu- 
lin to  the  usual  climatic,  hygienic,  and  open-air  method." 

The  use  of  tuberculin  as  a  means  of  establishing  the 
diagnosis  of  tuberculosis  in  cows,  supposed  to  be  infect- 
ed, has  proved  to  be  of  considerable  value,  and  it  is  now 
extensively  employed  for  this  purpose.  Eber,  in  a  sum- 
mary of  the  results  reported  by  various  authors  up  to 
the  15th  of  March,  1892,  gives  the  following  figures  :  In 
446  tuberculous  animals  inoculated  a  positive  reaction 
was  obtained  in  378  (84.75  per  cent.).  The  dose  given 
for  a  medium-sized  animal  was  usually  0.4  to  0.5  c.c.  of 
tuberculin  diluted  with  nine  to  ten  times  its  volume  of 
carbolic  acid  water  (0.5  per  cent.).  The  injection  was 
made  in  the  side  of  the  neck,  preferably  early  in  the 
morning  or  late  in  the  evening.  The  characteristic  reac- 
tion commenced  in  from  six  to  eighteen  hours  after  the 
inoculation,  and  lasted  from  three  to  twelve  hours.  The 
author  named  accepts  an  elevation  from  the  normal  tem- 
perature of  0.5°  C,  or  more,  lasting  for  several  hours,  as 
satisfactory  evidence  that  the  inoculated  animal  is  in- 
fected with  tuberculosis. 


TUBERCULOSIS.  287 

Johne  summarizes  the  reports  of  various  veterinarians 
in  Germany,  made  during-  the  year  1892.  The  total  num- 
ber of  animals  was  287,  the  dose  of  tuberculin  adminis- 
tered, from  0.1  to  0.5  gm.  Of  the  inoculated  animals  140 
reacted  and  were  slaughtered  ;  of  these  121  were  found  at 
the  autopsy  to  be  tuberculous,  and  19  were  not ;  17  re- 
acted, but  were  not  slaughtered ;  87  did  not  react,  but 
were  slaughtered,  of  these  82  proved  not  to  be  tubercu 
lous,  and  5  (all  reported  by  Schiitz,  Rockl,  and  Lydtin) 
were  tuberculous. 

Siedamgrotzky  (1892)  reports  the  results  obtained  in 
inoculations  practised  upon  259  cattle.  Of  these  209  re- 
acted with  a  temperature  of  40°  j3.,  or  more,  17  with  a 
temperature  of  39.5°  to  40°  C,  37  with  less  than  39.5°  C. 
In  197  the  temperature  reaction  amounted  to  1.5°  C,  or 
more,  in  8  it  was  between  1  and  1.5°  C,  and  in  54  less 
than  1°  C.  Those  in  the  last  g-roup  were  considered  as 
free  from  tuberculosis,  and  the  others  as  probably  tuber- 
culous (197  as  "very  x^robably  "). 

The  results  above  referred  to  considered  in  connection 
with  other  published  reports  indicate  that  a  reaction  of 
V  C,  or  more,  is  a  pretty  reliable  indication  that  the  in- 
oculated animal  is  tuberculous,  althoug-h  not  an  infallible 
test. 

Set'um-th€7'apy. 

Tizzoni  and  Cantanni  (1892)  have  made  experiments 
which  lead  them  to  the  conclusion  that  the  blood-serum 
of  immunized  guinea-pigs  contains  an  antitoxin  which 
may  be  successfully  used  in  the  treatment  of  infected 
animals  of  the  same  species,  but  their  experiments  can- 
not be  accepted  as  conclusive  and  require  confirmation, 


288  IMMUNITY   AND   SERUM-THERAPY. 

especially  in  view  of  the  fact  that  so  many  bacteriologists 
have  reported  their  failure  to  establish  immunity  in  these 
animals. 

Bernheim  (1894),  after  immunizing  animals  by  the  injec- 
tion of  sterilized  cultures  of  the  tubercle  bacillus  (treated 
for  an  hour  and  a  half  at  60°  C.  and  then  filtered),  used 
blood-serum  from  these  animals  in  the  treatment  of  tuber- 
culosis in  man.  From  1  to  3  c.c.  of  this  serum  was  in- 
jected every  second  day  between  the  shoulders.  The 
treatment  in  some  cases  was  continued  for  five  or  six 
months.  While  the  treatment  did  not  jorove  to  be  spe- 
cific, Bernheim  considers  the  results  obtained  somewhat 
encouraging-. 

Quite  recently  (1895)  Paquin  has  published  a  report  of 
his  method  of  obtaining  an  antitoxic  serum  and  the  re- 
sults of  treatment  in  a  number  of  cases  of  pulmonary 
tuberculosis.  He  obtains  his  serum  from  horses  immu- 
nized by  "  laboratory  processes  "  not  described  in  detail. 
His  conclusions  are  stated  as  follows  : 

"  1.  Sero-therapy  in  tuberculosis  has  proved,  so  far, 
efficacious. 

"  2.  Blood  -  serum  of  horses  seems  naturally  antago- 
nistic to  the  germs  of  tuberculosis,  but  cannot  in  its  nat- 
ural state  serve  in  treatment  with  much  good,  as  it  is  too 
slow  and  it  takes  enormous  quantities  of  it,  too,  to  pro- 
duce useful  results. 

"  3.  Horse  blood-serum  may  be  rendered  more  strongly 
antagonistic  to  the  tubercle  germ  by  the  treatment  of  the 
animals  by  a  proper  technique. 

"  4.  A  horse  treated  properly  three  months  may  yield 
serum  with  immunizing  power  that  will  jjrobably  prove 
sufficient  to  arrest  consumption  in  the  first  stages  in  three 
or  four  months,  and  sometimes  less  ;  and  in  the  second 
stage  in  four  to  six  months  or  a  year. 


TUBERCULOSIS.  289 

"  5.  Discrimination  should  be  made  in  the  selection 
of  cases,  and  judgment  exercised  to  exclude  from  the 
favorable  class  all  those  seriously  complicated  bacteri- 
olog'ically,  certain  cases  complicated  otherwise  patho- 
logically, and  other  patients  which  practice  alone  will 
suggest. 

"  6.  Inasmuch  as  hospitals  for  consumptives  are  needetl 
any  way,  the  success  has  been  sufficient  already  to  war- 
rant the  hope  that  in  the  near  future  such  institutions  shall 
be  built  on  modern  principles  of  hygiene,  for  the  exclu- 
sive treatment,  of  tuberculosis.  Sero-therapy,  with  the 
adjunct  treatments,  promises  better  success  than  ever  ob- 
tained before.  I  do  not  designate  this  serum  as  a  cure 
or  a  specific,  but  a  valuable  new  remedy." 


BIBLIOGRAPHY. 

Bernheim,  S.  :  Die  Behaiidlung  der  Tuberkulose  mit  immunisiertem 
serum.     Centralbl.  f.  Bakteriol.,  Bd.  XV.,  p.  654,  1894. 

Braucaccio  e  Solaro  :  Osservazioni  terapeutiche  sul  siero  di  sangue  di 
caue  nelle  tuberculosi  polmonare  incurabili.  Napoli,  1893,  p.  377, 
401. 

Bujwid,  O.  :  La  tuberculine,  sa  preparation,  etc.  Arch,  des  Sci. 
Biolog.,  St.  Petersbourg,  1892,  p.  243. 

Czaplewski,  E.,  uud  Roloff,  F.:  Beitrage  zur  Kenntniss  der  Tuberkii- 
linwirkung  bei  der  Experimentellea  Tuberkulose  der  Kaninchen 
und  Meerschweincben.     Berl.  klin.  Wchscbr.,  1892,  No.  29. 

Degive,  A.  :  Le  diagnose  de  la  morve  et  de  la  tuberculose  par  les  injec- 
tions hypodermiques  de  malleine  et  de  tuberculine.  Ann.  de  Med. 
veterin.,  xli.,  247,  1892. 

De  Scliweinitz  :  The  Attenuated  Bacillus  Tuberculosis  ;  its  Use  in  Pro- 
ducing Immunity  to  Tuberculosis  in  Guiuea-pigs.  The  Med.  News, 
Philadelphia,  1894  (Dec.  8). 

Diem  :  Versuche  mit  Tuberculin  bei  Huhnertuberkulose.  Monatssch. 
f.  Prakt.  Thierheilk.,  111,481,  1892. 

Escherich,  Th.  :  Die  Resultate  der  Koch'schen  Injectionen  bei  Scrophu- 
lose  und  Tuberkulose  des  Kinderalters.  Jahrb.  f.  Kinderheilk., 
XXXIII.,  369,  1892. 

Graraatschikoff,  A.  :  Ueber  die  Wirkung  des  Koch'schen  Mittels  auf 
Tuberkulose  Kaninchen.     Arbeit  a.  d.  Pathol.  •  Anatom.  Inst,  zu 
Tflbingen,  1892,  Heft  3. 
19 


290  IMMUNITY   AND   SERU3I-TIIERAPY. 

Grancher  et  Martin  :  Etude  sur  hi  vaccination  tuherculeuse.     Rev.  de 

la  Tuberculose,  1893,  p.  289. 
Hammerschlag:  Bakteriologisch-chepiische  Uiitersuclmugen  der  Tuber- 

ivelbacillen.    Sitzungsbericlit  der  K.  Akad.  der  Wissensch.  in  Wien. , 

Dec.  13,  1888. 
Hericonrt,  J.,  et  Ricliet,  Ch.  :  De  la  vaccination  coutre  la  tuberculose 

liumaine  par  la  tuberculose  aviaire.     Etudes  exper.  et  Clin,    sur 

la  Tuberculose,  iii.,  365,  1892. 
Influence  sur  I'infection  tuberculeuse  de  la  transfusion  du  sang 

des  chieus  vaccines  centre  la  tuberculose.      Compte-rendu,  Acad. 

des  Sci.,  cxiv.,  842,  1892. 
La  Vaccination  tuberculeuse  chez  le  cbien.     Le   Bull.   Med., 


1892,  pp.  741,  966. 
Helman,  C. :  Des  proprietes  de   la   tuberculiue  provenant  de   bacilles 

tuberculeux  cultives  sur  pommes  de  terres.     Arcb.  des  Sci.  Biolog. 

St.  Petersbourg,  t.  1,  p.  140. 
Johne,  A.  :  Tabellarische   Zusamnienstellung  der  im  J.  1892  mit  Tu- 

berkujiu  zu  diaguostischeu  Zwecken   angestellten   Impfversuche. 

Baumgarten's  Jahresbericht,  1892,  p.  676. 
Kaatzer,  P.  :    Ueber     das    Koch'sclie     Heilverfabren.     Dtsclie.    med. 

Wcbsckr.,1891. 
Kitasato,  S.  :   Ueber  die  Tuberculin — Bebandlung  tuberculoser  Meer- 

schweinchen.     Zeitschr.  f.  H3giene,  XII.,  321,  1892. 
Klebs,   E.  :    Die  Bebandlung    der   Tuberculose  niit    Tuberculocidin. 

Hamburg,  1892  (Voss). 
Klein,   H.  :    Ursacben  der  Tuberculinwirkung.     Bakteriologisch  -  ex- 

perimeutelle  Untersuchungen,  8%  107  p.,  Wien  u.  Leipzig  (Brau- 

maller),  1893. 
Koch  :    Weitere  Mittheilung  fiber    das   Tuberculin.     Deutsche  med. 

Woch.,  1891,  No.  43. 
Paquin,  P.  :  Anti-tubercle  Serum.     Journ.  Am.  Med.  Assn.,  vol.  xxiv., 

1895,  p.  341. 
Pfuhl,  E.  :  Beitrag  zur  Bebandlung  tuberkuloser  Meerscbweinchen  mit 

Tuberculinum  Kochii.  Zeitschr.  fur  Hygiene,  Bd.  XL,  1892,  p.  241. 
Sattler,  H.  :  Ueber  die  Wirkung  des  Tuberculins  auf  die  experimen- 

telle  Tuberkulose  beim  Kaninclien.     Dtsche.  med.  Wchschr.,  1891, 

Nos.  1  u.  2. 
Schiess-Bey  und  Kartulis  :     Ueber  die  Resultate  von  48  mit  Tuberkulin 

behandelten  Tuberkulosen.     Zeitsch.  fiir  Hygiene,  Bd.  XV.,  p.  229. 
Siedamgrotzky,  O.  :    Tuberkulinimpfungen  grosserer  Riuderbestande. 

Bericht.  tlber  d.  Veterinarwesen  i.  Krg.  Sachsen,  1891,  p.  228. 
Tizzoni,  G.,  und  Cantanni,  E.  :  Ueber  das  Vorhandensein  eines  gegen 

die  Tuberculose  Immunisirenden  Princeps  im  Blnte  von  Tbieren 

welche  nach  der  Metbode  von  Koch  behandelt  worden  siud.    Cen- 

tralbl.  f.  Bacteriol.,  XL,  82,  1893. 


TUBERCULOSIS.  291 

Trudeau,  E.  L.  :  Ej^e  Tuberculosis  and  Antitubercular  Inoculation  in 
the  Rabbit.     New  York  Medical  Journal,  1893,  vol.  ii.,  p.  97. 

Yamagiva  :  Versucbi'esultate  ilber  die  Wirkung  des  Tuberkulins  auf 
die  Impftuberkulose  des  Meerschweinchens  und  Kauinchens.  Vir- 
chow's  Archiv,  CXXIX.,  Heft  2,  1892. 


XXI. 

TYPHOID  FEVER. 

The  bacillus  discovered  independently  by  Koch  and 
by  Eberth  (1881)  and  carefully  studied  by  Gaffky  (1884)  is 
now  generally  recognized  as  the  specific  cause  of  typhoid 
fever.  It  is  constantly  found  in  the  form  of  small,  scat- 
tered colonies,  in  the  spleen,  the  liver,  the  glands  of  the 
mesentery,  and  the  diseased  intestinal  glands  in  fatal 
cases  of  typhoid  fever,  and  may  be  obtained,  by  puncture, 
from  the  spleen  during  life.  The  typhoid  bacillus 
closely  resembles  the  common  bacillus  found  in  the  in- 
testine of  healthy  individuals  which  was  first  described 
by  Escherich — Bacillus  coli  coTnmunis.  But  it  can  be 
differentiated  from  this  bj^  certain  biological  tests  and  is 
generallj'  recognized  by  bacteriologists  as  a  distinct 
species.  Both  the  typhoid  bacillus  and  the  "  colon  ba- 
cillus," however,  vary  considerably  as  a  result  of  condi- 
tions relating  to  their  environment,  and  it  is  difficult  to 
determine  whether  certain  bacilli  of  this  group,  some- 
times found  in  river-water,  etc.,  are  to  be  regarded  as 
varieties  of  one  or  the  other  of  the  species  mentioned  or 
as  distinct  species. 

Brieger  (1885)  found  in  cultures  of  the  typhoid  bacillus 
small  amounts  of  volatile  fat  acids,  and  when  grape-sugar 
has  been  added  to  the  culture  medium  lactic  acid.  He 
also  obtained  a  highly  alkaline  basic  substance  pos- 
sessing   toxic    i^roperties   which   he   named    typhotoxin 


TYPHOID   FEVER.  293 

(C,H„NOJ.  This  he  supposes  to  be  the  specific  product 
to  which  the  pathogenic  action  of  the  bacillus  is  due.  It 
produces  in  mice  and  guinea-pigs  salivation,  paralysis, 
dilated  pupils,  diarrhoea,  and  death. 

More  recent  experiments  by  Pfeilfer  (1894)  lead  him  to 
conclude  that  the  specific  poison  of  the  typhoid  bacillus 
is  not  present  in  filtered  cultures,  but  is  closely  associ- 
ated with  the  bacterial  cells.  According  to  Pfeifier  the 
bacillus  may  be  killed  by  a  temperature  of  54°  C.  without 
injury  to  this  toxic  substance.  The  fatal  dose  of  the 
dead  bacilli  is  from  3  to  4  milligr.  per  100  gm.  of  body- 
weight,  for  guinea-pigs.  Susceptible  animals  may  be 
immunized  by  means  of  this  toxic  substance,  and  their 
blood  is  found  to  contain  an  antitoxin  which  has  a  spe- 
cific bactericidal  action  upon  the  typhoid  bacillus.  But, 
according  to  Pfeilfer,  the  blood-serum  of  animals  immu- 
nized in  this  way  does  not  differ  from  normal  serum  in 
its  action  on  bacillus  coli  communis  and  other  species  of 
bacteria.  These  results  are  believed,  by  the  author  re- 
ferred to,  to  settle  the  question  of  the  specific  character 
of  the  typhoid  bacillus,  and  to  differentiate  it  from  nearly 
allied  species.  The  presence  of  a  typhoid  antitoxin  in 
the  blood-serum  of  individuals  who  have  recently  suffered 
an  attack  of  typhoid  fever  has  also  been  demonstrated 
by  Pfeiffer. 

Chantemesse  and  Widal  (1888)  first  showed  by  exj^eri- 
ment  that  susceptible  animals  could  be  made  immune 
against  the  pathogenic  action  of  this  bacillus  by  the  sub- 
cutaneous injection  of  sterilized  cultures.  Having  found 
that  four  drops  of  a  bouillon  culture,  three  days  old,  in- 
jected into  the  j^eritoneal  cavity  of  white  mice  caused  the 
death  of  these  animals  within  thirty-six  hours,  they  pro- 
ceeded to  inject   small   quantities    (I  c.c.)    of  a  culture 


294  IMMUNITY    AND   SERUM-THERAPY. 

which  had.  beeu  sterilized  by  heat,  and  found  that  after 
several  such  protective  inoculations  the  mice  no  longer 
succumbed  to  infection  by  an  unsterilized  culture. 

In  experiments  made  upon  rabbits,  Bitter  (1892)  ar- 
rived at  the  conclusion  that  the  immunity  which  he  pro- 
duced in  these  animals  by  the  intravenous  injection  of 
concentrated  sterilized  (by  filtration)  cultures  was  due  to 
the  presence  of  an  antitoxin  in  the  blood  of  the  immune 
animals.  Having-  found  that  control  animals  were  killed 
by  intravenous  injections  of  1  c.c.  of  his  concentrated  so- 
lution of  the  products  of  the  typhoid  bacillus,  he  added 
to  twice  this  amount  of  the  toxic  solution  a  certain  quan- 
tity (?)  of  blood-serum  from  an  immune  rabbit,  and  in- 
jected the  mixture  into  the  circulation  of  rabbits  with  a 
negative  result.  Control  experiments  in  which  the  toxic 
solution  was  mixed  with  blood-serum  from  non-immune 
animals  showed  that  this  had  no  antitoxic  effect,  and  the 
animals  died.  Bruschettini  obtained  (1892)  similar  re- 
sults in  his  experiments  upon  rabbits  with  cultures  ster- 
ilized by  heat  (60°  C).  He  concludes  from  his  experi- 
ments that  the  blood-serum  of  rabbits  immunized  in  this 
way  not  only  possesses  antitoxic  properties,  but  that  it 
has  g-reater  g-ermicidal  potency  for  the  typhoid  bacillus 
than  the  blood  of  normal  rabbits. 

Stern  (1892)  has  made  experiments  to  determine 
whether  the  blood  of  recent  convalescents  from  tj^phoid 
has  greater  germicidal  power  for  the  typhoid  bacillus 
than  that  of  other  individuals.  The  result  showed  that 
the  blood-serum  from  persons  who  had  recently  recov- 
ered from  typhoid  fever  had  no  increased  g"ermicidal 
power,  but  rather  showed  diminished  potenc\'  for  the  de- 
struction of  typhoid  bacilli.  But  blood  from  a  man  who 
had  suffered  an  attack  seventeen  and  a  half  years  pre- 


TYPHOID    FEVER.  295 

vioiisly  was  found  to  liave  unusual  bactericidal  power, 
altlioug-h  it  did  not  protect  white  mice  from  typhoid  in- 
fection. On  the  other  hand,  blood  from  recent  convales- 
cents served  to  immunize  white  mice,  thus  indicating  the 
presence  of  an  antitoxin.  This  is  also  shown  by  the  ex- 
periments of  Chantemesse  and  Widal  (1892),  who  report 
their  success  in  immunizing  suscejitible  animals  by  in- 
jecting the  blood-serum  of  other  animals  previously 
made  immune  by  repeated  injections  of  sterilized  (by 
heat)  cultures.  The  authors  last  named  have  also  tested 
the  blood-serum  of  typhoid-fever  patients,  of  recent  con- 
valescents from  the  disease,  and  of  persons  who  had 
suffered  an  attack  some  years  before  the  experiment  was 
made.  The  experiments  were  made  upon  guinea-pigs. 
Tlie  authors  conclude  that  "  in  general  the  guinea-pig  is 
immunized  against  the  action  of  virulent  typhoid  cult- 
ures by  the  subcutaneous  injection  of  a  small  quantity  of 
serum  of  persons  who  have  suffered  an  attack  of  the  dis- 
ease, no  matter  how  remote."  But  this  immunity  was 
shown  to  be  of  short  duration,  and  quite  different  from 
that  induced  by  the  injection  of  sterilized  cultures,  which 
does  not  immediately  follow  the  introduction  of  the  toxic 
substances,  but  requires  a  certain  number  of  days  for  its 
development.  The  degree  of  immunity  is  said  by  the 
authors  last  named  to  depend  to  a  considerable  extent 
upon  the  dose  given,  and  the  animals  treated  in  this  way 
still  resisted  virvilent  cultures  at  the  end  of  two  months. 
On  the  other  hand,  injections  of  blood-serum  from  im- 
mune individuals  were  effective  in  doses  of  a  single  c.c, 
within  a  few  hours,  and  the  immunity  conferred  had  a 
comparatively  brief  duration. 


296  IMMUNITY    AND   SEKUM-THERAPY. 

Seru7n-therapy. 

lu  their  experiments  iu  the  treatment  of  infected  rab- 
bits and  guinea-pigs  with  serum  from  immune  animals 
Chantemesse  and  Widal  (1892)  obtained  favorable  results 
when  injections  of  2  c.c.  were  made  into  the  cavity  of  the 
abdomen  within  a  few  hours  after  infection.  Favorable 
results  were  also  obtained  by  injecting  in  the  same  way 
blood-serum  from  individuals  who  had  suffered  a  recent 
or  remote  attack  of  the  disease.  Animals  which  recovered 
were  immune  against  infection  by  the  bacillus,  but  had 
no  increased  resistance  against  the  toxic  action  of  filtered 
cultures.  In  two  cases  in  which  typhoid  patients  in  the 
eleventh  and  thirteenth  day  of  the  disease  were  treated 
with  considerable  doses  of  serum  from  immune  animals, 
the  progress  of  the  disease  was  not  arrested. 

Neisser  (1893)  by  the  injection  of  serum  from  a  typhoid 
convalescent,  fourteen  days  after  the  termination  of  the 
fever,  was  able  to  protect  mice  from  three  or  four  times 
the  lethal  dose  of  tyiihoid  cultures.  He  injected  1  or  2 
c.c.  daily  for  four  days. 

Stern  (1894)  has  made  similar  and  more  numerous  ex- 
periments upon  mice  and  guinea-pigs,  to  test  the  anti- 
toxic power  of  the  serum  of  individuals  who  had  suffered 
an  attack  of  typhoid  fever.  The  inoculations  were  made 
in  the  peritoneal  cavity  in  the  proportion  of  from  1  to 
1  to  10  to  1  of  serum  and  culture.  In  guinea  -  pigs 
the  serum  was  injected  from  sixteen  to  twenty -four  hours 
before  infection.  Positive  results  were  obtained  with 
serum  from  6  out  of  8  recent  convalescents,  and  with  3 
out  of  5  individuals  who  had  suffered  an  attack  from  one 
to  ten  years  previously  ;  and  negative  results  with  serum 
from  two  persons  who  had  suffered  'attacks  more  than 


TYPHOID    FEVER.  297 

ten  j'^ears  before  the  experiment  was  made.  In  a  series 
of  experiments  with  blood-serum  from  fatal  cases  of 
typhoid  this  was  found  to  be  still  more  potent  than  the 
serum  of  recent  convalescents.  In  doses  of  from  0.02  to 
0.05  c.c.  it  protected  guinea-pig's  from  lethal  doses  of  a 
virulent  typhoid  culture  injected  after  an  interval  of 
twenty  hours.  The  blood  was  obtained  from  a  vein  an 
hour  after  the  death  of  the  patient.  In  a  series  of  com- 
parative experiments  with  blood  from  individuals  who 
had  never  had  an  attack  of  tyi3hoid  a  protective  influence 
was  shown  to  be  exercised  in  some  cases,  but  larger 
doses  were  required.  Stern  does  not  attempt  to  explain 
this  fact,  but  suggests  the  possibility  that  persons  whose 
blood-serum  showed  this  antitoxic  power  may  have  at 
some  time  suffered  a  mild  and  unrecognized  attack  of 
typhoid  fever. 

Cesaris-Demel  and  Orlandi  (1894)  have  obtained  results 
which  confirm  those  above  referred  to  as  regards  the  pro- 
tective and  therapeutic  value  of  serum  from  animals  im- 
munized against  the  typhoid  bacillus  ;  and  have  obtained 
similar  results  in  experiments  with  bacillus  coli  commu- 
nis. Moreover,  they  conclude,  as  a  result  of  their  exijeri- 
mental  researches,  that  animals  which  have  been  im- 
munized for  the  "  colon  bacillus  "  furnish  a  serum  which 
has  protective  and  therapeutic  value  against  infection  by 
the  tyiahoid  bacillus,  and  the  reverse.  The  serum  from 
immunized  animals  has  been  used  by  the  bacteriologists 
referred  to  in  the  treatment  of  typhoid  fever  in  man,  and 
the  results  are  thought  to  be  favorable,  although  the 
number  of  cases  so  treated  is  still  too  small  to  justify  any 
definite  conclusion. 

We  may  refer  briefly,  before  concluding  the  present 
chapter,  to  recent  experiments  in  the  treatment  of  typhoid 


298  IMMUNITY    AND   SKRUM-TIIERAPY. 

fever  with  sterilized  cultures  of  the  typhoid  bacillus 
g-rowii  in  thymus  bouillon  (Friinkel)  and  of  bacillus  pyo- 
cyaneus  (Rumpf).  Frankel  and  Manchot  (1893)  treated 
fifty-seven  cases  of  typhoid  fever  in  the  new  general  hos- 
pital at  Hamburg-Eppendorf  with  cultures  of  the  typhoid 
bacillus  in  thymus  bouillon,  Avhicli  had  grown  for  three 
days  in  an  incubating  oven  at  37"  C,  and  were  then 
sterilized  by  exposure  to  a  temperature  of  60°  C.  The 
treatment  was  commenced  by  the  subcutaneous  injection 
of  0.5  c.c.  of  the  sterilized  culture ;  the  following  day  a 
dose  of  1  c.c.  was  given ;  after  this  the  injection  was  re- 
peated every  second  day,  and  the  dose  was  increased  each 
time  by  1  c.c.  Usually  there  was  a  noticeable  rise  of 
temperature  as  a  result  of  the  injection,  and  in  some  cases 
a  more  or  less  jaronounced  chill.  On  the  third  day  of 
treatment  there  was  usually  a  fall  in  the  temperature, 
followed  by  a  still  greater  reduction  on  the  following- 
day.  The  conclusion  is  reached  that  the  course  of  the 
fever  is  materially  influenced  by  the  treatment,  that  it  is 
changed  from  a  continued  to  a  more  or  less  remittent 
character,  and  that  complete  apyrexia  occurs  in  a  shorter 
time. 

Rumpf  (1893)  was  induced  to  test  the  value  of  steril- 
ized cultures  of  Bacillus  jDyocyaneus,  because  it  had  been 
shown  by  experiments  upon  animals  that  such  sterilized 
cultures  exercised  an  immunizing  influence  against  ty- 
phoid infection.  He  followed  Friinkel's  method,  giving 
on  the  first  day  0.5  c.c.  of  the  sterilized  culture,  on  the 
second  day  1  c.c,  on  the  fourth  day  2  c.c,  the  sixth  day  4 
c.c,  the  eighth  day  6  c.c.  The  injections  were  made  in 
the  gluteal  region.  They  were  commonly  followed  by  a 
rise  in  the  temperature  and  a  subsequent  fall,  with  dimin- 
ished pulse-rate  and  profuse  perspiration.     Sometimes  a 


TYPHOID   FEVER.  299 

chill  and  a  rise  of  temperature  followed  the  injection. 
No  effect  was  observed  upon  the  secretion  of  urine  or  the 
respiration,  but,  as  a  rule,  a  general  improvement  in  the 
feeling  of  the  iDatient  and  a  prompt  disappearance  of  de- 
lirium, when  it  existed,  was  noted.  Thirty  cases  were 
treated,  with  two  deaths,  one  from  pneumonia  and  one 
from  intestinal  hemorrhage. 

In  a  recent  communication  (1894)  Kraus  and  Buswell 
have  reported  the  results  of  additional  experiments. 
They  treated  in  all  twelve  cases  with  sterilized  pyocya- 
neus  cultures  ;  ten  recovered,  and  two  died.  As  a  rule, 
the  immediate  result  of  an  injection  was  a  reduction  of 
temperature  lasting  for  several  hoiirs.  It  was  found  also 
that  an  increase  in  the  number  of  leucocytes  in  the  blood 
followed  the  injections,  and  the  same  result  occurred 
when  the  injections  of  sterilized  pyocyaneus  cultures 
were  made  in  rabbits.  The  conclusion  is  reached,  how- 
ever, that  this  method  of  treatment  has  no  specific  cura- 
tive value. 

BIBLIOGRAPHY. 

Bitter,  H.  :  Ueber  Festigung  von  Versuchsthieren  gegen  die  Toxine 
der  Typhusbacillen.     Zeitschr.  f.  Hygiene,  XII.,  298,  1892. 

Cesaris-Demel  »nd  Oiiandi  :  Die  Serunitherapie  und  das  Bacterium 
Coli.     Abst.  in  Centralbl.  fur  Bakteriol.,  XVI.,  246,  1894. 

Chantemesse  et  Widal  :  De  Timmunite  contre  le  virus  de  la  fievre  ty- 
phoide  conferee  par  les  substances  solubles.  Ann.  de  I'lnst.  Pas- 
teur, t.  ii.,  1888,  p.  54. 

Etude  experinientale  sur  I'exaltation,  Timmunisation.  et  la  thera- 

peutique  de  I'infection  typhique.  Annal.  de  I'lnst.  Pasteur,  1892, 
p.  755. 

Frankel,  E.  :  Ueber  spezitisclie  Bebandlung  des  Abdominallyphus. 
Dtscli.  nied.  Wchschr.,  1893,  No.  41. 

Kraus,  Fr. ,  u.  Buswell,  H.  C.  :  TJeber  die  Bebandlung  des  Typbus  ab- 
doniinalis  mit  abgetoteten  Pyocyauus  -  Kulturen.  Wien.  Win. 
Wcbscbr.,  1894,  p.  511,  595. 


300  IMMUNITY    AND   SEIIUM-THERAPY. 

Montcfusco,  H.  :  Contributo  alia  l)iologia  del  bacillo  del  tifo.     La  Rif. 

Med.,  1893,  p.  155. 
Neisser  :  Zeitscbr.  fUr  klin.  Med.,  1893,  Bd.  XXII. 
Petruschky,  J.  :  Ueber  die  Ait  der  Patbogenen  Wirkung  des  Typbiis- 

bacillus  a\rf  Tbiere  und  Uber  die  Verleibung  des  Impfscbutzes 

gegen  dieselbe.     Zeitscbr.  f.  Hygiene,  XII.,  261,  1892. 
Pfeiffer,  R.  :  Ueber  die  specifiscbe  Immunitatsreaction  der  Typbu.sba- 

cillen.     Vorlaufige  Mittbeilung.     Deutscbe  raed.  Wcbscbr.,  1894, 

No.  48,  p.  898. 
Riimpf,  Tb.  :  Die  Bebandlung  des  Typbus  xVbdominalis  mit  Abgetote- 

ten  Kulturen  des  Bac.  Pyocyaneus.     Dtscbe.  med.  Wcbscbr.,  1893, 

p.  987. 
Stern,  R.  :  Ueber  Immunitat  gegen  Abdominaltyplms.     Dtscbe.  nied. 

Wcbscbr.,  1892,  No.  37. 
Ueber  die  Wirkung  des  Menscblicben  Blutserums  auf  die  Expe- 

rimeutelle  Typbusiat'ectiou.     Zeitscbr.  fUr  Hyg.,  XVI.,  458, 1894. 


xxn. 

YELLOAV   FEVER. 

In  the  writer's  report  (1890)  upon  the  results  of  his  in- 
vestigations relating-  to  the  etiology  of  yellow  fever,  his 
conclusions  are  stated  as  follows  : 

"  The  experimental  data  recorded  in  this  report  show 
that — 

"  The  specific  infectious  agent  m  yellow  fever  has  not 
been  demonstrated.  The  most  approved  bacteriological 
methods  fail  to  demonstrate  the  constant  presence  of  any 
particular  microorganism  in  the  blood  and  tissues  of 
yellow-fever  cadavers. 

"  The  microorganisms  which  are  sometimes  obtained  in 
cultures  from  the  blood  and  tissues  are  present  in  com- 
paratively small  numbers,  and  the  one  most  frequently 
found  {Bacterimn  coli  communis)  is  present  in  the  intes- 
tine of  healthy  individuals,  and  consequently  its  occa- 
sional presence  cannot  have  any  etiological  import. 

"  A  few  scattered  bacilli  are  present  in  the  liver,  and 
probably  in  other  organs,  at  the  moment  of  death.  This 
is  shown  by  preserving  portions  of  liver,  obtained  at  a 
recent  autopsy,  in  an  antiseptic  wrapping. 

"  At  the  end  of  twenty -four  to  forty-eight  hours  the  in- 
terior of  a  piece  of  liver  so  preserved  contains  a  large 
number  of  bacilli  of  various  species,  the  most  abundant 
being  those  heretofore  mentioned  as  occasionally  found 
in  the  fresh  liver-tissue,  viz.,  BaderiuTn  coli  comirmnis  and 
Bacxllxis  cadaveris. 

"  Blood,  urine,  and  crushed  liver-tissue  obtained  from  a 


302  IMMUNITY   AND   SERUM-THERAPY. 

recent  autopsj'  are  not  patliog-enic,  in  moderate  amounts, 
for  rabbits  or  guinea-pigs. 

"  Having-  failed  to  demonstrate  the  presence  of  a  specific 
'  germ  '  in  the  blood  and  tissues,  it  seems  probable  that 
it  is  to  be  found  in  the  alimentary  canal,  as  is  the  case  in 
cholera.  But  the  extended  rtesearches  made  and  recorded 
in  the  present  report  show  that  the  contents  of  the  intes- 
tines of  yellow-fever  cases  contain  a  great  variety  of  ba- 
cilli and  not  a  nearly  pure  culture  of  a  single  species,  as 
is  the  case  in  recent  and  tyj)ical  cases  of  cholera." 

At  the  time  of  my  visit  to  Brazil  (1887)  Dr.  Domingos 
Freire  iDresented  me  with  a  culture  of  a  micrococcus 
which  he,  at  that  time,  considered  the  specific  yellow- 
fever  germ.  This  micrococcus  did  not  at  all  conform 
with  the  descriptions  which  he  had  repeatedly  published 
of  his  "  cryptococcus  xanthogenicus."  It  did  not  pro- 
duce pigment,  either  black  or  yellow,  was  not  pathogenic 
for  small  animals,  except  when  injected  in  very  large 
doses,  and  gave  rise  to  no  symptoms  in  inoculated  ani- 
mals which  could  be  identified  with  those  of  yellow 
fever.  On  the  contrary,  it  was  an  ordinary  stapholycoc- 
cus,  which  corresponded  in  its  morphology  and  growth 
in  culture  media  with  the  well-known  staphyloccus  pyo- 
genes albus.  With  reference  to  this  micrococcus  I  quote 
from  my  report  above  referred  to  as  follows  : 

"  That  this  micrococcus  bears  no  relation  to  the  etiol- 
ogy of  yellow  fever  is  fully  proved  by  my  extended  cult- 
ure experiments  in  Havana  during  the  summers  of  1888 
and  1889.  In  the  entire  series  of  autopsies  I  have  made 
cultures  from  the  liver,  and  in  a  considerable  number 
from  blood  obtained  directly  from  the  heart,  and  I  have 
not  obtained  this  micrococcus  of  Freire  in  a  single  in- 
stance, although  the  culture  medium  commonly  employed 
— flesh  peptone-gelatine — is  a  very  favorable  one  for  the 


YELLOW   FEVER.  303 

growth  of  this  coccus.  Nor  has  it  been  found  iu  the  ex- 
tended series  of  sections  which  I  have  made  from  the 
liver  and  kidney  preserved  in  alcohol  from  my  Havana 
autopsies.  In  one  case,  only  (Case  X.,  1888)  I  have  found 
micrococci  in  sections  from  the  kidney,  but  as  the  micro- 
coccus of  Friere,  in  its  form  and  dimensions,  resembles 
many  others,  it  is  impossible  to  say  that  this  is  or  not  the 
Freire  coccus.  The  finding-  of  micrococci  in  this  case, 
however,  does  not  invalidate  the  general  result,  which  is 
that  micrococci  are  not  found  hi  tJie  Mood  and  tissues  of  yel- 
low fever  cadavers.'''' 

In  a  paper  read  at  the  Quarantine  Conference,  held  in 
Montg-omery,   Ala.,  in   March,  1889,    the   present   writer 

says : 

"  I  may  say  before  g-oing-  any  further  that  my  faith  in  a 
living-  infectious  agent  as  the  specific  cause  of  this  dis- 
ease is  by  no  means  diminished  by  my  failure  thus  far  to 
demonstrate  the  exact  form  and  nature  of  this  lij  potheti- 
cal  '  g-erm.'  The  present  state  of  knowledge  with  refer- 
ence to  the  etiolog-y  of  infectious  diseases  in  g'eneral,  and 
well  kno^vn  facts  relating  to  the  origin  and  spread  of  yel- 
low-fever epidemics  fully  justifies  such  a  belief.  The  a 
priori  grounds  for  such  faith  I  stated  as  long  ago  as  1873, 
in  a  paper  published  in  the  American  Journal  of  the  3£edi- 
ccd  Sciences  (July,  1873) ;  and  the  progress  of  knowledge 
since  that  date  has  all  been  in  the  direction  of  support- 
ing this  a  2^riori  reasoning.  But  yelloAv  fever  is  by  no  ' 
means  the  only  infectious  disease  in  which  satisfactory 
evidence  of  the  existence  of  a  living  infectious  agent  is 
still  wanting.  In  the  eruptive  fevers  generally  no  dem- 
onstration has  been  made  of  the  specific  etiological 
agent — at  least  none  which  has  been  accepted  by  compe- 
tent pathologists  and  bacteriologists.  Again,  in  the  in- 
fectious disease  of  cattle  known  as  pleuro-pneumonia, 
notwithstanding  very  extended  researches  by  competent 
investigators  in  various  parts  of  the  world,  no  satisfac- 


304  IMMUNITY    AND   SERUM-THEEAPY. 

tory  demonstration  of  the  germ  lias  been  made.  Tlie 
same  is  true  of  hydrophobia,  in  which  disease  we  are 
able  to  say  with  confidence  the  infectious  agent  is  pres- 
ent in  the  brain  and  spinal  cord  of  animals  which  suc- 
cumb to  rabies  ;  this  infectious  agent  is  destroyed  by 
a  temperature  which  is  fatal  to  known  pathogenic 
microorganisms  (65°  C),  and  by  various  germicidal 
agents,  yet  all  efforts  to  cultivate  it  or  to  demonstrate  its 
presence  in  the  infectious  material  by  staining  processes 
and  microscopical  examination  have  thus  far  been  un- 
successful." 

In  a  paper  read  before  the  College  of  Physicians  in 
Philadelphia,  in  1888,  I  give  the  following  account  of  my 
investigations  with  reference  to  the  inoculations  practised 
by  Dr.  Domingos  Freire  in  Brazil : 

"  Facts  relating  to  the  endemic  and  epidemic  preva- 
lence of  yellow  fever,  considered  in  connection  with  the 
present  state  of  knowledge  concerning  the  etiology  of 
other  infectious  diseases,  justify  the  belief  that  yellow 
fever  is  due  to  a  living  microorganism,  capable  of  devel- 
opment, under  favorable  local  and  meteorological  con- 
ditions, external  to  the  human  body,  and  of  establishing 
new  centres  of  infection  when  transported  to  distant  local- 
ities. 

"  Inasmuch  as  a  single  attack  of  yellow  fever,  however 
mild,  protects,  as  a  rule,  from  future  attacks,  there  is 
reason  to  hope  that  similar  protection  would  result  if  a 
method  could  be  discovered  of  inducing  a  mild  attack  of 
the  disease  by  inoculation  or  otherwise.     .     .     . 

"  My  own  researches,  recorded  in  the  foregoing  report, 
show  that  no  such  microorganism  as  Dr.  Domingos 
Freire,  of  Brazil,  has  described  in  his  published  works, 
or  as  he  presented  to  me  as  his  yellow-fever  germ  at  the 
time  of  my  visit  to  Brazil,  is  found,  as  he  asserts,  in  the 
blood  and  tissues  of  typical  cases  of  yellow  fever. 

"  There  is  no  satisfactory  evidence  that  the  method  of 


YELLOW    FEVER.  305 

inoculation  practised  by  Dr.  Domingos  Freire  has  any 
prophylactic  value. 

"Dr.  Freire' s  Protective  Inoculations. — Having  demon- 
strated that  Dr.  Freire's  claim  to  have  discovered  the 
specific  cause  of  yellow  fever  is  without  scientific  founda- 
tion, it  may  be  thought  that  no  further  demonstration  is 
required  in  order  to  show  that  his  x^reventive  inoculations 
are  without  value  ;  for  these  inoculations  are  said  to  have 
been  made  with  cultures  containing  the  attenuated  mi- 
crobe of  yellow  fever.  These  inoculations  have,  however, 
been  made  upon  so  large  a  scale,  and  the  statistical  re- 
sults, as  presented  by  Dr.  Freire,  appear  so  favorable  to 
his  method  that  it  becomes  necessary  to  analyze  these 
statistics :  and  if,  as  he  claims,  they  establish  the  fact 
that  the  mortality  from  yellow  fever  is  very  much  less 
among  those  who  have  been  inoculated  by  him  than 
among  non-inoculated  persons  exposed  in  the  same  way, 
we  shall  be  obliged  to  concede  the  value  of  his  method, 
although  the  rationale  of  the  protective  influence  may 
not  be  apparent.  In  my  detailed  report  I  have  reviewed 
at  length  Dr.  Freire's  statistics  in  the  light  of  the  facts 
developed  by  my  personal  researches  in  the  city  of  Rio 
de  Janeiro,  where  the  inoculations  were  made.  I  cannot 
attempt  to  bring  the  evidence  before  j^ou  at  the  present 
time,  and  I  have  already  stated  to  you  my  conclusion 
with  reference  to  the  matter.  In  support  of  this  conclu- 
sion I  shall,  however,  quote  a  few  extracts  from  my  report. 

"In  1884  Dr.  Freire  inoculated  418  persons,  Avhose 
names,  ages,  place  of  residence,  and  length  of  residence 
in  Brazil  are  given  in  an  appendix  to  his  principal  work, 
published  in  1885.  In  regard  to  the  evidence  afforded  by 
these  inoculations,  I  have  written  as  follows  : 

"  Dr.  Freire  admits  that  '  during  the  epidemic  season  a 
great  number  of  the  vaccinated  were  attacked  by  the  mal- 
ady,' but  claims  that  these  attacks  were  of  a  mild  char- 
acter, yet  he  gives  us  the  names  of  seven  vaccinated  per- 
sons who  died  from  the  disease.  This  list  has  been 
added  to  by  some  of  Dr.  Freire's  confreres,  as  will  be  seen 
20 


306  IMMUNITY    AND   SERUM-THERAPY. 

by  the  following-  translation  of  a  letter  published  in  one  of 
the  newspapers  of  liio,  and  bearing-  date,  May  5,  1887. 
This  letter  is  sig-ned  by  Dr.  Aran  jo  Goes,  at  present  a 
member  of  the  Central  Board  of  Health,  and  a  g-entleman 
whose  statements  are  worthy  of  the  fullest  confidence  : 

"  '  My  letter  to  the  Imperial  Academy  of  Medicine  hav- 
ing been  published,  it  now  behooves  me  to  publish  the 
statistics  relating  to  the  vaccinations  on  Morro  de  Yiuva. 

"  '  One  fact  seems  to  me  to  be  definitely  demonstrated, 
that  is  the  icorthlessness  of  Dr.  Freire's  vaccinaiion,  as  is 
well  known  to  the  medical  profession  of  this  city. 

"  '  A  year  ago  I  wrote  the  following  : 

"  '  The  want  of  skill  which  he  displayed  in  his  first  ex- 
periments, the  false  conclusions  which  he  has  drawn 
therefrom,  and  the  tliougiitless  precipitation  with  which 
he  has  hastened  to  make  known  incomplete  results  with- 
out accompanjdng  them  with  a  single  qualifying:  remark 
vitiate  all  the  methods  to  wdiicli  he  may  hereafter  resoi-t 
to  corroborate  his  statements  {Journal  do  Coinmerc'io, 
April  20,  1883). 

" '  The  mortality  among  the  persons  vaccinated  on 
Morro  da  Viuva  furnishes  one  more  proof  that  I  was 
rig-ht  in  saying-  this,  as  I  now  proceed  to  demonstrate, 

" '  There  were  vaccinated  in  this  district  60  persons. 

"  '  Sixteen  removed  shortly  after  the  commencement  of 
the  epidemic,  and  44  remained  exposed  to  its  influence. 
Of  these  22  had  yellow  fever,  9  of  whom  died.     .     .     .' 

"  In  1885  Dr.  Freire  resumed  his  inoculations  on  a 
larger  scale-,  but  instead  of  selecting  unacclimated 
strangers,  those  inoculated  w^ere  for  the  most  part  natives 
of  Brazil,  or  Portuguese  who  had  lived  for  a  number  of 
years  in  Rio  and  who  had  passed  through  one  or  more 
epidemics.  A  considerable  number  of  negroes  were  also 
inoculated  and  included  in  the  statistical  tables.  With 
reference  to  Dr.  Freire's  statistics  for  the  year  1885  I 
quote  from  my  report  as  follows  : 

"  '  Dr.  Freire  has  omitted  to  state  one  very  important 
fact  with  reference  to  the  vaccinations  practised  during 


YELLOAY    FEVER.  307 

the  period  included  in  this  tabular  statement.  The  date 
of  the  vaccinations  is  not  given.  Fortunately  I  am  able 
to  supply  this  omission  from  his  journal  containing-  the 
names  of  the  vaccinated,  which  he  kindly  placed  in  my 
hands  during  my  stay  in  Rio.  I  find  from  this  record  that 
the  inoculations  were  practised  as  follows  : 

January 392 

February 342 

March 611 

April 189 

May 273 

June 813 

July 481 

"  '  Now  it  is  well  known  that  June  and  July  are  months 
during  which  3^ellow  fever  does  not  jarevail  in  Eio,  and 
that,  in  fact,  the  month  of  May  furnishes,  as  a  rule,  but 
few  cases. 

"  '  The  exposure  even  in  an  epidemic  year  amounts  to 
very  little  during  the  months  of  May,  June,  and  July,  and 
may  be  considered  practically  nil  in  a  year  like  1885, 
when  the  whole  mortality  was  only  278  in  a  city  of  400,- 
000  inhabitants.  But  Dr.  Freire  has  included  in  his  list 
1,294  persons  who  were  vaccinated  during-  the  healthy 
winter  months  of  June  and  July,  and  who  presumably 
had  been  exposed  during-  the  preceding  comparatively 
unhealthy  months  of  January,  February,  March,  and 
April.  If  these  1,291  individuals  were  protected  from  an 
attack  of  yellow  fever  by  the  inoculation  practised  in 
June  or  July,  what  protected  them  from  being-  attacked 
during-  the  preceding  months  when  yellow  fever  was  pre- 
vailing to  some  extent  ?     .     .     .' 

"  I  pass  now  to  the  year  1886,  during-  which  Dr.  Freire 
inoculated  2,763  Brazilians  and  710  foreig-ners,  again  in- 
cluding in  his  statistical  tables  those  vaccinated  after  the 
epidemic  season  had  passed.  In  reviewing-  these  statis- 
tics I  remark  as  follows  : 

"  '  We  have  quoted  this  last  report  of  Dr.  Freire  in  ex- 
tenso  in  order  to  do  him  full  justice  by  allowing-  him  to 


308  IMMUNITY   AND   SERUM-THERAPY. 

state  his  own  case.  We  shall  now  proceed  to  show  that 
his  statistics  are  fallacious,  and  that  the  percentage  of 
mortality  among  the  vaccinated,  which  he  finds  to  be  ten 
times  less  than  among  the  non-vaccinated,  results  from  a 
misuse  of  the  statistical  method  and  from  a  number  of 
factors  which  are  favorable  to  Dr.  Freire's  statistics  as 
he  has  stated  them,  but  not  to  a  fair  test  of  his  method 
of  prophylaxis. 

"  '  In  the  first  place,  we  would  call  attent/on  to  the  fact 
that  while  during-  the  comparatively  healthy  year,  1885, 
the  immunity  among  the  vaccinated  of  that  year  is  said 
to  be  complete  (see  report  of  1885),  the  number  of  deaths 
during  the  epidemic  year  which  followed  is  stated  by  Dr. 
Freire  himself  to  have  been  eight.  Taking  all  of  the 
vaccinated  of  the  two  years,  and  without  making  any 
allowance  for  the  considerable  number  of  persons  vacci- 
nated who  had,  no  doubt,  left  the  city  before  the  epidemic 
of  1886  occurred,  Dr.  Freire,  with  a  total  of  6,524  vacci- 
nated, and  a  total  of  8  deaths,  makes  the  proportion  one 
per  thousand.  This  is  equivalent  at  the  outset  to  an  ad- 
dition of  1,476  persons  to  the  number  vaccinated,  who 
being  imaginary  persons  and  not  having  been  exposed  to 
the  epidemic  influence  simply  aid  in  rounding  up  the 
general  percentage  of  mortality  in  Dr.  Freire's  favor  to 
the  even  figure  of  one  per  thousand.  This  is  but  one  of 
many  factors  which  go  to  make  this  favorable  showing. 
Reference  to  Dr.  Freire's  MS.  journals,  which  he  kindly 
placed  in  my  hands,  shows  that  of  the  total  number  vac- 
cinated during  the  two  years,  4,465  were  vaccinated 
prior  to  the  epidemic  of  1886  ;  that  is  to  say,  before  the 
1st  of  January,  1886.  How  many  of  these  left  the  city 
before  the  outbreak  of  the  epidemic,  how  manj'^  were 
only  temporarily  in  the  city  when  vaccinated,  how  many 
died  from  other  diseases  I  cannot  say ;  but  it  is  a  signifi- 
cant fact  that  of  the  3,051  vaccinated  prior  to  August, 

1885,  Dr.  Freire  has  only  one  fatal  case  to  report,  while 
out  of  460  persons  vaccinated  in  January  and  February, 

1886,  he  reports  5  deaths,  a  mortality  of  more  than  one 


YELLOAY    FEVER.  309 

per  cent.,  wliicli  he  gives  as  tlie  general  mortality  among 
the  non-vacciuated.  This  is  not  apparent  from  his  own 
statement  of  the  case,  but  is  nevertheless  true,  as  I  shall 
proceed  to  show.  In  his  report,  which  we  have  just 
given  in  full,  he  does  not  give  the  date  of  the  vaccination 
of  these  individuals,  but  upon  referring  to  his  MS.  jour- 
nals for  1886  I  find  that  No.  3  of  his  list,  Jose,  son  of 
Jose  da  Costa  Vieira,  was  vaccinated  February  12,  1886  ; 
No.  4,  Paschoal  Ruffino,  on  the  6th  of  February,  1886  ; 
No.  5,  Henri  Constance,  on  the  1st  of  January,  1886  ;  No, 
6,  Fernando  Argenteiro,  on  the  20tli  of  February,  1886  ; 
and  No.  7,  Antonio  Saraiva,  on  the  12th  of  February,  1886. 
The  same  MS.  record  for  1886  shows  that  during  these 
two  months — January  and  February,  1886 — the  total  num- 
ber vaccinated  by  Dr.  Freire  was  460.  That  is  to  saj;-, 
the  mortality  among  those  vaccinated  during  these  two 
months  was  more  than  one  per  cent.  On  referring  to  the 
mortality  list  of  the  city  for  the  same  two  mouths  I  find 
the  total  number  of  deaths  to  have  been  369,  which  in  a 
total  susceptible  population  of  160,000  (Dr.  Freire's  esti- 
mate) would  give  a  mortality  of  1  in  436.' " 

Dr.  Carl  Seidl,  of  Rio  de  Janeiro,  wlio  is  Director  of 
the  San  Sebastian  Yellow  Fever  Hospital  in  that  city,  in 
a  recently  (1894)  published  communication  calls  attention 
to  the  fact  that  Dr.  Freire's  yellow-fever  vaccinations 
"  have  been  in  progress  since  1883  and  yelloAv  fever  con- 
tinues, becoming  year  by  year  more  formidable  and  de- 
structive, instead  of  being  graduallj'^  stamped  out  as  was 
prophesied  by  Freire.  Statistics  will  never  convince  the 
bacteriologists  of  the  world,  the  medical  fraternity  of 
Brazil,  nor  the  public,  of  the  value  of  these  vaccinations. 
For  where  is  the  physician,  where  is  the  intelligent  man 
in  this  town,  who  does  not  know  cases  and  cases  in  which 
said  vaccination  was  not  only  useless  but  perhaps  harm- 
ful because  it  caused  a  false  confidence  ?     In  the  labora- 


810  IMMUNITY    AND    SEKUM-TIIERAPY. 

fcories  of  Paris,  Vienna,  and  Berlin  are  long-  series  of 
pathogenic  germs,  some  only  recently  announced,  yet 
there  is  no  culture  of  the  xanthococcus  which  has  been 
discovered  for  so  many  years  and  i^ut  to  practical  account 
in  Kio."  * 

The  evidence  in  favor  of  the  value  of  the  protective  in- 
oculations practised  by  the  method  proposed  by  Profes- 
sor Carmona  (1881),  of  the  City  of  Mexico,  is  not  satisfac- 
tory, and  this  method  was  soon  abandoned.  It  consisted 
in  the  subcutaneous  injection  of  material  obtained  by 
desiccating  the  urine  of  yellow-fever  patients  freely  ex- 
posed to  the  air  in  shallow  vessels.  Carmona  says,  with 
reference  to  his  inoculations  : 

"  I  usually  place  one  or  two  centigrammes  of  the  drj' 
residue  in  a  gramme  of  distilled  water,  I  triturate  it  in 
such  a  manner  that  the  mixture  is  as  perfect  as  possible, 
and  charging  a  Pravaz  syringe,  I  make  a  subcutaneous 
injection  in  the  right  arm.  The  results  are  various,  but 
no  serious  accident  has  ever  occurred. 

"  I  count,  to-day,  nearly  two  hundred  persons  inocu- 
lated, and  among  them  several  experienced,  some  hours 
after  the  inoculation,  a  febrile  movement,  which  some- 
times caused  the  thermometer  to  mount  to  38.5°  C.  The 
duration  of  this  febrile  movement  did  not  exceed  twenty- 
four  hours.  The  local  accidents  have  been  most  varied. 
There  was  almost  always  tumefaction  at  the  point  of  in- 
oculation, but  the  extent  and  size  of  this  tumefaction 
varied  greatly.  In  many  cases  there  was  redness  of  the 
skin.  These  local  phenomena  lasted  four  or  five  days,  but, 
in  general,  those  inoculated  continued  about  their  ordina- 
ry affairs.  Once  only  I  have  seen  developed  a  phlegmon, 
which  terminated  by  sui3puration." 


*  Quoted  from  an  editorial  article  in  the  Journal  of  the  American  Medical  As- 
sociation of  January  5,  1895. 


YELLOW   FEVEK.  311 

The  "  mosquito  inoculations  "  of  Dr.  Carlos  Finlay,  of 
Havana,  have  not  g-iven  results  which  have  induced  any 
of  his  medical  confreres  to  adopt  his  method ;  and  the 
facts,  as  stated  by  himself,  do  not  appear  to  give  any 
support  to  the  supposition  that  these  inoculations  have  a 
protective  value.  The  method  consists  in  allowing-  a  mos- 
quito to  fill  itself  with  blood  from  a  yellow-fever  patient, 
and,  after  two  or  three  days,  when  it  has  digested  this 
meal,  applying  it  to  the  susceptible  individual,  who  is 
supposed  to  be  inoculated  when  the  insect  introduces  its 
sting  for  the  purpose  of  filling-  itself  with  blood.  But 
there  seems  slig-ht  chance  that  such  inoculation  would 
occur  even  if  the  infectious  element  had  been  proved  to 
be  present  in  the  blood  drawn  from  the  yellow-fever  pa- 
tient. For  this  blood  is  dig-ested  and  passes  through  the 
alimentary  canal,  and  the  lancet  and  sucking"  apparatus 
of  the  insect  would  probably  be  pretty  well  cleaned  dur- 
ing- the  interval  between  its  application  to  the  yellow- 
fever  case  and  the  person  to  be  protected.  Dr.  Finlay 
says  in  his  last  published  paper  (1894)  : 

"  In  my  inoculation  experiments  the  interval  between 
the  application  of  the  contaminated  mosquito  to  a  suscep- 
tible person  and  the  appearance  of  the  first  symptoms  of 
a  mild  attack  of  the  disease  (when  such  a  one  did  occur) 
has  varied  between  five  and  twenty-five  days,  the  latter 
term  being  the  one  that  I  have  fixed  upon,  beyond  which 
any  morbid  symptoms  would  be  considered  as  indepen- 
dent of  the  inoculation.  In  the  majority  of  cases  no 
pathogenic  effects  were  produced." 

It  is  well  established  that  the  period  of  incubation  in 
yellow  fever  is  short,  usually,  if  not  always,  less  than  five 
days  ;  and  there  is  not  the  slightest  reason  for  the  assump- 
tion that  the  so-called  mosquito  inoculation  had  anything 


812  IMMUNITY    AND   SERUM-THERAPY. 

to  do  with  the  "  acclimation  fever  "  which  a  small  propor- 
tion of  those  inoculated  sufltered  in  from  five  to  twenty - 
five  daj'^s  after  this  operation.  All  of  the  inoculated 
remained  in  Havana  and  were  exposed  to  the  same  in- 
fiuences  which  produce  the  so-called  "  acclimation  fever" 
in  other  individuals  not  inoculated. 
Dr.  Finlay  states  his  results  as  follow's  : 

"  Among"  the  eighty-seven  who  have  been  under  obser- 
vation the  following-  results  have  been  recorded  : 

"  1.  Within  a  term  of  days,  varying"  between  five  and 
twenty-five  after  the  inoculation,  one  presented  a  mild  al- 
buminuric attack,  and  tldrteen  only  'acclimation  fevers." 

"  2.  Among"  the  seventy-three  who  did  not  present  an 3" 
distinct  attack  within  the  first  twenty-five  days — 
40  were  subsequently  attacked  with  simple  '  acclimation 
fever.' 
5  suffered    an   attack    of    regular    albuminuric    yellow 

fever. 
4  had  melano-albuminuric  yellow  fever. 
24  have  never  had  any  fever  of  the  yellow-fever  type. 

73 

"  Tioo  of  the  melano-albuminuric,  and  one  of  the  albu- 
minuric, were  fatal,  giving"  a  mortality  of  3.87,  equivalent 
to  3|  per  cent.  One  occurred  in  1884,  and  the  two 
others  in  1893." 

The  most  significant  fact  in  this  statement  is  that  nine 
of  those  inoculated  suffered  attacks  of  yellow  fever- 
three  of  which  were  fatal.  The  fact  that  twenty-four  out 
of  the  eighty-seven  inoculated  have  remained  in  Havana 
without  suffering  an  attack  of  yellow  fever  cannot  be  ac- 
cepted as  evidence  that  they  are  protected  by  the  mos 
quito  inoculation  practised  by  Dr.  Finlay,  for  it  is  well 


YELLOW   FEVER.  313 

known  in  HaNtuia  that  strangers  wlio  have  not  been  inoc- 
ulated often  remain  in  that  city  for  many  years  without 
having-  yellow  fever.  Moreover,  the  forty  cases  of  "  ac- 
climation fever,"  which,  according  to  Dr.  Finlay,  occurred 
among  the  inoculated,  belong  in  the  same  category  as 
the  thirteen  cases  which  occurred  in  from  five  to  twenty- 
live  days  after  the  inoculation  was  practised.  This 
twenty-five-day  limit  is  entirelj^  arbitrary,  and  it  is  diffi- 
cult to  see  how  Dr.  Finlay  can  attribute  the  so-called 
"  acclimation  fever,"  which  occurs  in  less  than  twenty- 
five  days,  to  the  direct  effects  of  his  mosquito  inocula- 
tions, and  cases  which  occiir  after  twenty-five  days  to  a 
protective  influence  exercised  by  these  inoculations. 

As  a  matter  of  historical  interest  in  connection  with 
the  subject  of  protective  inoculations  against  yellow 
fever,  I  introduce  here  an  account  of  the  inoculations 
made  in  1854  and  1855  by  Dr.  William  Lambert  de  Hum- 
boldt, and  those  made  in  1864  by  Drs.  Lebredo  and  Cis- 
neros,  of  Havana. 

Dr.  de  Humboldt  claimed  to  be  a  nephew  of  the  cele- 
brated Alexander  v.  Humboldt.  He  asserted  that  he  had 
discovered  a  sure  means  of  protecting  from  yellow  f-ever 
by  inoculations  with  the  venom  of  a  poisonous  snake, 
found  in  Mexico,  but  kept  as  a  secret  the  precise  species 
of  snake  from  which  his  material  for  inoculations  was  ob- 
tained. His  theory  was  based  upon  the  fact  that  symp- 
toms somewhat  resembling  those  of  yellow  fever  are  pro- 
duced by  the  venom  of  certain  poisonous  snakes.  These 
sj^mptoms  are  hemorrhage  from  the  gums,  slow  pulse, 
fever,  etc.  The  first  experiments  of  Humboldt  were 
made  in  Vera  Cruz,  in  1847,  by  authority  of  the  govern- 
ment, upon  condemned  prisoners.  According  to  Boudin 
the   matter  inoculated   was  an   ounce   of   sheep's   liver 


314  IMMUNITY  "and   SEllUM-TIIERAPY. 

wliicli  had  been  bitten  l>y  six  of  these  ijoisonous  ser- 
pents. This  was  left  to  undergo  putrefaction  before  it 
was  used  for  the  inoculations. 

A  history  of  Humboklt's  inoculations  has  been  written 
by  Dr.  Nicolas  B.  L.  Manzini.  This  is  a  volume  of  240 
pages,  which  was  published  in  Paris  in  1858.  I  shall 
quote  from  this  work,  which  is  entitled  "  Histoire  de 
f inoculation  2^^'eservatiue  de  la  Jievre  jaune,  prat'tqnee  pur 
ordre  du  gouvemement  espagnol,  a  Vhopital  Jiul'datre  de  la 
Havane.  Hedlgce  par  Nicolas  B.  L.  Manzini,  docteur  en 
medecine  de  la,  Facidte  de  Paris,  mernhre  titulaire  de  la 
Societe  mediccde  d' emulation  de  Paris,  medecin  de  V Associ- 
ation de  hienfaisarvce  frangaise  de  la  Havane.'" 

"  I.  In  the  month  of  October,  1854,  Dr.  William  Lambert 
de  Humboldt,  residing  then  in  New  Orleans,  wrote  to 
General  Don  Jose  de  la  Concha,  Governor  of  the  island 
of  Cuba,  announcing  to  him  that  he  had  discovered  a  sub- 
stance, the  active  principle  of  which  was  the  venom  of  an 
ophidian,  which  substance,  inoculated  by  vaccination  in 
persons  who  were  strangers  in  the  localities  where  j^ellow 
fever  reigns  as  an  epidemic,  protected  them  from  this 
terrible  malady.  M.  de  Humboldt  said  that  during  a 
period  of  nine  years  he  had  inoculated  fourteen  hundred 
and  hftj^-eight  individuals.  Of  those  whose  history  he 
had  been  able  to  follow  he  had  only  seen  seven  attacked 
with  yellow  fever,  and  of  these  only  two  had  died.  Out 
of  three  hundred  and  eighty-six  inoculated  in  New  Or- 
leans he  had  not  in  any  case  seen  yellow  fever  character- 
ized by  the  pathognomonic  symptoms — black  vomit,  etc. 
Finally,  M.  de  Humboldt  offered  to  apply  his  preserva- 
tive to  the  Spanish  troops  of  the  island  of  Cuba,  in  a 
most  disinterested  manner. 

"II.  General  Concha  first  consulted  Dr.  Basterreche, 
chief  of  the  corps  of  military  sanitation  of  the  island  of 
Cuba,  with  reference  to  this  important  affair,  who  consid- 


YELLOW   FEVER.  315 

ered  it  prudent  to  consult  with  some  of  his  medical 
friends,  who  all  g-ave  a  favorable  opinion.  Then  General 
Ooncha,  who  at  the  outset  took  a  lively  interest  in  the 
question,  consulted,  officially,  the  University,  submitting- 
at  the  same  time  a  memoir  by  M.  de  Humboldt  relative 
to  it.  This  corporation  judged  that  the  experiment  Avas 
admissible  and  that  the  facts  would  decide  the  question. 
In  consequence  of  this  decision  M.  de  Humboldt  was 
invited  to  come  to  Havana.  A  ward  in  the  military  hos- 
pital w^as  placed  under  his  absolute  direction.  It  was 
likewise  agreed  that  all  of  the  inoculated  who  should  sub- 
sequently fall  sick  should  be  placed  in  his  care,  and  that 
he  could  be  assisted  or  reiDlaced  by  persons  of  his  selec- 
tion. A  commission  of  tire  University  was  named  to  fol- 
low the  march  of  the  operations  and  to  make  exact  obser- 
vations. It  Avas  composed  of  Drs.  Cowley,  Castroverde, 
and  Benjnmeda. 

"  III.  M.  de  Humboldt  had  scarcely  arrived  in  Havana 
Avhen  I  put  myself  in  relations  with  him.  He  appears  to 
lie  thirty-five  to  thirty-six  years  old.  He  is  blond,  high, 
and  slender.  His  chest,  flattened  in  front,  offers  the  con- 
formation peculiar  to  the  tuberculous.  .  .  .  Although 
his  health  is  precarious  he  is  endowed  with  a  feverish 
activity  and  with  a  resistance  to  work  which  is  surpris- 
ing. M.  de  Humboldt  speaks  four  languag-es,  among 
them  the  Spanish  and  French,  as  perfectly  as  is  possible 
for  a  foreigner.  German  is  his  native  tong-ue,  and  he 
speaks,  besides,  English.     .     .     . 

"  The  special  history  of  the  phenomena  of  the  inocula- 
tion which  we  are  about  to  write  is  derived  from  seventy- 
nine  observations  collected  and  written  out  by  M.  de 
Humboldt  and  myself.  Besides,  we  have  drawn  some  in- 
formation relative  to  the  circulation  and  headache  from 
one  hundred  and  thirteen  other  cases,  collected  in  the 
military  hospital  under  the  direction  of  M.  de  Humboldt. 

"  We  have  little  to  say  of  the  character  of  the  substance 
inoculated,  which  has  precisely  the  appearance  and  the 
odor  of  the  liquid  residue  of  animal  putrefaction. 


316  IMMUNITY   AND   SERUM-THERAPY. 


1.    LOCAL   PHENOMENA   OF   THE   INOCITLATION. 

"  As  soon  as  the  iiiocnlatioii  is  made,  a  crossed  bandage 
is  applied  to  the  puncture  and  no  further  attention  is 
paid  to  it.  If  it  is  examined  at  the  end  of  a  few  moments 
it  will  be  found  to  be  surrounded  with  an  elevation  in  the 
form  of  a  white  papule,  diaphanous,  and  quite  analogous 
to  that  produced  by  the  puncture  of  a  bed-bug.  This 
phenomenon  was  visible  at  the  end  of  five  minutes  or 
even  of  three.  It  was  no  longer  seen  at  the  end  of  twenty- 
four  hours,  of  twelve,  or  even  of  ten.  A  sensation  of  tin- 
gling and  numbness,  a  veritable  phenomenon  of  slight  an- 
esthesia, soon  manifested  itself  in  the  forearm  and  lasted 
for  a  variable  time  ;  we  have  seen  it  joersist  until  the 
fourth  day  in  the  case  of  Madame  Mercedes  Parodi.  We 
have  never  seen  any  swelling  of  the  axillary  glands. 

2.    OUTLINE   OF  THE   SICKNESS   INDUCED. 

"  Syncope  may  occur  at  the  moment  of  inoculation — this 
soon  i:)asses — or  a  nervous  trembling,  which  is  more  rare 
but  which  lasts  longer.  The  pulse  is  accelerated  under 
the  influence  of  the  emotion  of  the  moment.  At  the  end 
of  seven  hours  (all  of  the  times  which  follow  are  the 
mean  time  deduced  from  the  extremes)  the  pulse  is 
modified  in  a  permanent  manner ;  it  is  more  frequent  or 
slower,  stronger  or  more  feeble.  At  the  end  of  eleven 
hours  there  is  febrile  heat ;  at  the  end  of  fourteen,  head- 
ache, thirst,  loss  of  appetite  ;  at  the  end  of  sixteen  the 
face  is  red,  the  conjunctivae  injected,  laclirymation.  A 
commencement  of  swelling  of  the  gums  is  observed  and 
slight  colicky  pains  are  experienced,  produced  by  the 
medicine  which  the  patient  commenced  to  take  immedi- 
ately after  the  inoculation. 

"  First.  At  the  end  of  eighteen  hours  pain  in  the  gums, 
which  commence  to  be  colored  around  the  borders  of  the 
teeth ;  pain  in  the  salivary  glands  and  in  the  direction  of 
the  different  nervous  branches  of  the  face  and  cranium. 


YELLOW   FEVER.  317 

Second :  of  nineteen  hours,  pains  in  the  lower  jaw  in  the 
direction  of  the  inferior  maxillary  nerve,  lassitude  ;  of 
twenty  hours,  bitter  taste,  somnolence,  oedema  of  the  face ; 
of  twenty-two  hours,  feeling-  of  constriction  of  the  throat, 
without  visible  modification  of  the  mucous  membrane  ; 
of  twenty-three  hours,  yellowness  of  the  skin  ;  of  twenty- 
four  hours,  hemorrhag"e  from  the  gums  ;  of  twenty-eig-ht 
hours,  eyes  yellow,  chills ;  of  twenty-nine  hours,  inflam- 
mation of  tonsils  ;  of  thirty  hours,  pain  in  reg-ion  of 
kidneys  ;  of  thirty-six  hours,  oedema  of  eyelids ;  of  thirty- 
eight  hours,  muscular  and  articular  pains  ;  of  forty  hours, 
toothache  ;  of  seventy-two  hours,  oedema  of  the  lower  lip  ; 
at  different  times,  erotic  phenomena.  During  conva- 
lescence, cutaneous  itching ;  cutaneous  eruptions  of  dif- 
ferent kinds. 

3.    IMMEDIATE  TREATMENT. 

"  As  soon  as  the  inoculation  was  made  we  administered 
a  sirup  composed  as  follows  : 

^ .  Sirop.  de  mikania  giiaco 187  gm. 

Sirop.  de  rhubarbe 125  gm. 

lodure  de  potassium 4  gm. 

Gomme  gutte 12  gm. 

D.  et  M. 

"  This  sirup  was  administered  in  the  following-  manner  : 
First  day,  a  tablespoonful  every  two  hours  ;  second  day, 
one  every  four  hours ;  third  day,  one  morning-  and  even- 
ing. If  the  symptoms  are  more  violent,  the  interval  is 
shortened  ;  and  if  that  is  not  sufficient,  it  is  necessary  to 
add  to  the  sirup  an  infusion  of  mikania,  a  teacupful 
every  two  hours." 

We  shall  not  attempt  to  follow  our  author  in  his  ac- 
count of  the  symptoms  produced  by  the  poisons  of  the 
various  venomous  snakes  and  in  his  attempt  to  show  a 
resemblance  between  these  symptoms  and  those  of  yellow 
fever.     Nor  shall  we  quote  his  account  of  the  results  of 


318 


IMMUNITY   AND   SERT  M-TIIERAPY. 


the  inoculations  made,  inasmuch  as  we  have  these  from 
an  official  source  in  the  report  of  Dr.  Bastan-eche,  which, 
with  great  fairness,  Dr.  Manzini  has  included  in  his  vol- 
ume, although  he  insists,  contrary  to  the  opinion  and  fig- 
ures of  Dr.  Bastarreche,  that  the  inoculations  were  at- 
tended with  a  comparatively  favorable  result. 

It  will  be  sufficient  to  introduce  here  a  sing-le  one  of 
the  tables  appended  to  the  report  of  Dr.  Bastarreche. 


TABLE  NO.  V. — COMPARATrVE   SUMMARY. 


Not  IxoctXATED. 

ISOCCI-ATED. 

Ratio  of 

Attacked.      Died.      Ratio. 

Number. 

Attacked. 

Died. 

Murtality. 

Army 

Navy 

1,045 
264 

254     24.31 

47    i 17.80 

1 

1,214 
1,263 

84 
144 

21 
46 

25.00 
31.94 

Total.... 

1,309     !    301      22.99 

2,477 

228 

67 

29.39 

Nothing  further  has  been  heard  of  Dr.  Humboldt's 
method  of  inoculation  in  Havana,  and  the  inference  is 
that  by  common  consent  the  experiment  made  on  so  large 
a  scale,  and  under  such  favorable  conditions,  is  regarded 
as  having  demonstrated  its  inutility. 

Another  attempt  to  protect  from  yellow  fever  by  inocu- 
lations is  that  made  in  1864  by  two  physicians  of  Havana, 
members  of  the  Academy  of  Sciences,  and  recorded  in 
the  "  Anales  "  of  the  Academy.  "W'e  quote  the  translation 
which  Dr.  Stanford  E.  Chaille,  President  of  the  Havana 
Yellow  Fever  Commission  of  the  National  Board  of 
Health,  has  introduced  into  his  elaborate  and  valuable 
"  Keport :  "  * 


*  Annual  Report  of  the  Natioual  Board  of  Health  for  1880,  p.  160. 


YELLOW    FEVER.  319 

"  In  June,  1864,  Drs.  Lebredo  and  Cisneros,  members 
of  the  Academy,  and  distinguished  physicians,  tested  the 
prophylactic  value  of  inoculated  dew,  by  request  of  Drs. 
Masnata  and  Fraschieri,  who  had  claimed  for  it  protec- 
tive power. 

"  The  substance  used  was  not,  as  had  been  generally 
supposed,  natural  dew,  but  an  artificial  dew  obtained  by 
the  condensation  of  vapor  of  the  atmosphere  of  the  closed 
room  of  a  yellow-fever  patient,  and  collected  on  the  sur- 
face of  bottles  containing  water  of  a  lower  temperature 
than  that  of  the  surrounding-  air.  After  jirolonged  ex- 
amination the  following  were  our  conclusions  : 

"  Yellow  fever  is  not  a  contagious  nor  an  inoculable 
disease,  hence  the  inoculation  of  dew  cannot  be  effec- 
tive. There  is  no  such  entity  as  the  so-called  '  fever  of 
acclimation,'  audit  has  not  been  proved  that  the  ailments 
thiis  designated  protect  from  yellow  fever.  The  symp- 
toms following  the  inoculation  of  '  rocio  '  lack  the  uni- 
formity necessary  to  constitute  a  classifiable  pathologi- 
cal condition  as  dependent  solely  on  the  inoculation  ; 
the  very  slight  intensity  of  the  phenomena  discredit  their 
identity  with  those  of  the  so-called  '  fever  of  acclima- 
tion ; '  in  many  instances  no  phenomena  have  ensued, 
and  all  the  results  obtained  are  explicable  by  disregard  of 
hygienic  laws.  In  three  counter-exiDeriments  distilled 
water  was  inoculated.  In  one  case  more  remarkable 
results  ensued  than  in  any  case  inoculated  with  '  rocio,'  in 
a  second  the  results  wei-e  as  mild,  and  in  a  third  case  no 
results  at  all  ensued.  Finally,  as  the  result  of  experi- 
ments, the  inoculation  of  '  rocio '  is  ineffective,  and 
equally  as  negative  as  inoculations  of  black  and  of  bilious 
vomit." 

BIBLIOGRAPHY. 

Carraona  y  Valle  :  LeQons  sur  I'etiology  et  la  prophylaxie  de  la  flevre 
jaiiue.     Mexico,  1885. 

Fiiilay  :  Inoculations  for  Yellow  Fever  by  Means  of  Contaminated 
Mosquitoes.  American  Journal  of  the  Medical  Sciences,  Phila- 
delphia, 1891,  vol.  cii.,  p.  264. 


320  IMMUNITY    AND   SERUM-TIIEP.APY. 

Fiulay  :  Yellow  Fever.  Edinburgh  Medical  Journal,  July,  October,  and 
November,  1894. 

Freire  :  Doctrine  microbienne  de  la  tievre  jaune,  et  ses  inoculations  pre- 
ventives.    Rio  de  Janeiro,  1885,  630  pp. 

Resultats  obtenus  par  I'inoculatiou  preventive  du  virus  attenue 

de  la  fievre  jaune.     Paris,  1887. 

ISlernberg  :  Report  on  the  Etiology  and  Prevention  of  Y'ellow  Fever. 
Washington,  1891,  265  pp.,  21  plates. 

Report  upon  the  Prevention  of  Y'ellow  Fever  by  Inoculation. 

Washington,  1888. 

Dr.  Finlay's  Mosquito  Inoculations.     American  Journal  of  the 


Medical  Sciences,  Philadelphia,  1891,  p.  627. 


INDEX. 


Abuin,  immunity  from  toxic  ac- 
tion of,  53 
Acquired  immunity,  34-71 
Alexins,  28,  46 

Alkalinity  of  blood  in  immune  an- 
imals, 26 
Anthrax,  75-98 

albumose,  preparation  of,  89 
bacillus,  attenuation  of  viru- 
lence, 77,  87 
bacillus,  chemical  products  of, 

90,91 
cured  by  cultures  of  S.  pyo- 
genes, 243 
immunity  of  rats,  87 
inoculations,    Cenkowski's 

method,  92,  93 
inoculations,     Chauveau's 

method,  81,  82 
inoculations,   Pasteur's  meth- 
od, 80 
inoculations.   T  o  u  s  s  a  i  u  t '  s 

method,  76,  82 
inoculations,  priority  in  mak- 
ing, 75 
inoculations,  results  of,  85,  91, 

93 
serum-therap}^  95 
spores  not  formed  at  43^  C,  77 
susceptibility  to,  3 
Antidiphtherin,  Klebs's,  168 
Anlitoxins,  24 

characters  of,  46 
discovery  of,  46 
explanation  of  production  of, 

57-58 
in  milk  of  immune  animals, 

53,  159 
mode  of  action  of,  61 
Antitoxin  of  diphtheria,  results  of 
treatment  with,  163-168 
of  pneumonia,  220 
of  small-pox  (V),  231 


Antitoxin  of  serpent  venom,  60 

of  tetanus,  47,  258 

unit,  Behring's,  157 
Aronson,    method    of    producing 

diphtheria  immunity,  158 
Attenuation  of  virulence,  37-39 

Bacteria,  pathogenic,  in  healthy 
persons,  13 
role  of,  in  nature,  12 
sapropliytic,  13 
Behring,  method  of  estimating  im- 
munizing value,  156 
Black   leg,  See   Symptomatic   au- 

thrax 
Blood-serum,  germicidal  power  of, 
17,  20 
antitoxic  value  of,  156 
Buchner's  explanation  of   immu- 
nity, 25 

Chauveau's  niethod  of    produc- 
ing immunity,  82,  85 

explanation  of  immunity,  81 
Chemiotaxis,  16 
Chicken  cholera,  99-110 

cholera,    attenuation  of   viru- 
lence, 105 

cholera,  augmentation  of  viru- 
lence, 105 

cholera,  etiology  of,  104 

cholera,     identical    witli     Da- 
vaine's  septic;emia,  103 

cholera,  infection  by  feedinu', 
100 

cholera,  Kitt's  method  of  yixo- 
ducing  inmiunity,  109 

cholera,     protective     inocula- 
tions in,  104 

cholera,   results  of  protective 
inoculations,  108 

cholera,   susceptible    aninuds, 
99 


322 


INDEX. 


Chicken  cholera,  Toussaint's  meth- 
od of  inoculation,  107 
Cholera,  111-147 

aulitoxin,  in  milk,  16 
cultures  fatal  to  guinea-pigs, 

114 
cultures,  increase  in  virulence, 

115 
Ferran's     protective    inocula- 
tions, 123-130 
Haffkine's  protective  inocula- 
tions, iaO-140 
production  of  immnnitv  in  an- 
imals, 114-119 
prophylaxis,  144 
second  attacks  of,  120,  121 
serum-therapy,  141-143 
spirillum,  attenuation  of  viru- 
lence, 116 
spirillum,    toxic  products  of, 
111-114 
Cobra  venom,  neutralized  by  tet- 
anus antitoxin,  61 
Crotou-oil,  immunit}^  from  action 
of,  63 

Davaine's  septicaemia,  102 

Defensive  proteids,  23 

Diphtheria,  148-168 

antitoxin,  Arousnn's,  158 
antitoxin,  Behring's,  155-159 
antitoxin,  Roux's,  160-163 
antitoxin,  results  of  treatment 

with,  163-168 
bacillus,  attenuation  of,  150 
bacillus,  toxic  products  of,  153 
bacillus,  where  found,  151 
immunization  of  goats,  158 
immunization  of  horses,  161 
mortality,  statistics  of,  164 
natural  inimunit}^  of  rats  and 

mice,  151 
production  of  immunity.  154 
susceptible  animals.  149 
toxic  action  of  filtered  cultures, 
152 

Epidemics,  Pasteur's  explanation 

of,  78,  106 
Erysipelas,  immunity  from,  243 

Ferra^''s  method  of  inoculation 

against  cholera,  122-130 
Foot-and-mouth  disease,  172 
Fowl  cholera,   iSee  Chicken  chol- 
era 


GERMic'iDAr.   action    of   blood-se- 
rum, 21,  57 
Glanders.  174-181 

bacillus,    toxic    products    of, 

174-176 
diairnosis  of,  by  use  of  mal- 

leine,  178-180 
production  of  immunity  from, 

177 
serum-therapy,  180 
susceptibility  to,  3 
Globulin,    germicidal    action    of, 
33 

Haffkine's  method  of  inoculation 

against  cholera,  130-141 
Hankin's  method  of  producing  im- 
mnnitv, 89 
Hog  cholera,  182-185 

cholera,   bacillus,   toxic  prod- 
ucts of,  184 
cholera,     protective     inocula- 
tions in,  183,  184 
Hog  erysipelas,  186-190 

erysipelas,  protective  inocula- 
tions in,  186-188 
ervsipelas,    serum-therapy  in, 
188-190 
Hydrophobia,  192-206 

immunity    from    intravenous 

injections,  201 
immunit}',   Italian  method  of 

producing.  202 
immunity,    Pasteur's    method 

of  producing,  194,  196 
period  of  incubation,  195 
protective  inoculations  in  doc:s, 

195 
protective  inoculations,  inten- 
sive method,  198 
protective  inoculations,  results 

of,  198-201 
serum-therapy  in,  202-205 
virus,  where  found,  194-195 

Immunity,  See  also  Protective  in- 
oculations 

acquired.  34-71 

acquired,  author's  explanation 
of,  in  1881,  41 

acquired,  Bushner's  explana- 
tion of,  25 

acquired,  Metchnikoff's  ex- 
planation of,  16-20 

acquired,  bv  injection  of  fil- 
tered cultures,  40,  49,  293 


INDEX. 


323 


Immunity,  acquired,  from  anthrax 
infection,  80-95 

acquired,  from  cliolera  infec- 
tion, 114-141 

acquired,      from     diplillieria, 
infection,  154-163 

acquired,  from  glanders,  177- 
180 

acquired,  from  hydrophobia, 
195-202 

acquired,     from    septicaemia, 
238-239 

acquired,    from    pneumonia, 
318-223 

acquired,  from  small-pox,  230- 
232 

acquired,  from   streptococcus 
infection,  241-242 

acquired,  from   tetanus,  256- 
262 

acquired,    from    tuberculosis, 
278-283 

acquired,  from  typhoid  infec- 
tion, 293-295 

acquired,  from  vegetable  tox- 
albumins,  51-54 

acquired,  from  venom  of  ser- 
pents, 56 

natural,  3-33 

natural,  explanation  of,  14,  31 

natural,  neutralized  by  anaes- 
thetics, 10 

natural,  neutralized  by  chemi- 
cal substances,  6 

natural,     neutralized    by    fa- 
tigue, 9 

natural,  neutralized  bj^  inani- 
tion, 9 

natural,  neutralized  by  i)utie- 
factive  products,  8 

natural,  race,  explanation  of,  5 
Immunizing  value  of  blood-serum, 

156 
Infection,  predisposing  causes.  10 

influence  of  quantity  of  infec- 
tious ma-terial,  11 
Influenza,  207-208 

protective  inoculations  in  rab- 
bits, 207 
Influenza  in  horses,  209-210 


Klebs's  antidiphtherin,  168 
Koch's  discoverv  of  the  tubercle 
bacillus,  271 
mctiiod  of  preparing  tubercu- 
lin, 276 


Leucocytes,  increase  of,  in  infec- 
tious diseases,  30 

Mallein,    method   of   preparing, 
175-178 
use  of,  in  diagnosis  of  gland- 
ers, 178-180 
Metchnikoflf  theory  of  immunity, 

16-20 
^lilk,  antitoxins  in,  53 

diphtheria  antitoxin  in,  159 
tetanus  antitoxin  in,  261 
preparation  of  antitoxin  from, 
54 

NucLEiNS,  chemical  characters  of , 
30 
germicidal  power  of,  28 

Pasteur's  pioneer  researches,  77- 
81 
method   of   attenuating  viru- 
lence, 37 
Pathogenic  bacteria  in  healthy  in- 
dividuals, 13 
I  virulence,  causes  of  variation 

in,  11 
;  Phagocytosis,  14 

Pleuro-pneimionia  of  cattle,  211- 
I       215 
'  Pneumonia,  216-226 

immunity  from,  218-223 
micrococcus  of,  217 
predisposing  causes,  10 
serum-therapy  in,  224 
j  Protective  inoculations.   See    also 
\  Immunity 

I  inoculations,  results  of,  in  an- 

I  thrax,  91-95 

inoculations,     results    of,    in 
I  cholera,  120-141 

inoculations,  results  of,  in  hy- 

dropiiobia,  195-202 
inoculations,  results  of,  in  h(  g 

cholera,  183 
inoculations,  results  of,  in  hos 

erysipelas,  186-188 
inoculations,  results  of,  in  in- 
fluenza, 207 
inoculations,  results  of,  in  in- 
fluenza of  horses,  209 
inoculations,    results     of,     in 
pleuro-pneumonia  of  cattle, 
211-215 
inoeidations,    in     pneumonia, 
219-223 


324 


INDEX. 


Protective  inooiilations,  in  small- 
pox, 228-2cil 

iiKjcuhitioiis,  in  streptococcus 
infection,  241-244 

inoculations,  in  swiue  plague, 
238,  240 

inoculations,  in  symptomatic 
anllirax,  24G-2.'5() 

inoculations,  in  tetanus,  258- 
2G0 

inoculations,  in  typhoid  infec- 
tion, 29:J-295 

inoculations,  in  tuberculosis, 
278-283 

inoculations,  in  yellow  fever, 
Carmona's,  310 

inoculations,  in  yellow  fever, 
Finlay's,  311-313 

inoculations,  in  yellow  fever, 
Freire's,  305-309 

inoculations,  in  yellow  fever, 
Humboldt's,  3i3-318 

Rabbit  septicaemia,  237 

Rabies,  See  Hydrophobia 

Rattlesnake  venom,  immunity 
from,  56 

Rausch brand,  245-250 

Ricin,  immunity  from  toxic  action 
of,  52 

Rinderpest,  227 

Rotlilauf,  Sef.  Hog  erysipelas 

Rouget,  See  Hog  erysipelas 

Roux,  method  of  producing  anti- 
toxin of  diphtheria,  160-162 

SCHWEINESEUCHE,   237-240 

Second  attacks  in  infectious  dis- 
eases of  man,  35 
Serum-therapy,  in  anthrax,  95 
therapy,  in  cholera,  141-143 
therapy,    in    diphtheria,    163- 

168 
therapy,  in  glanders,  180-181 
therapvt    in    hog    erysipelas, 

188-190 
therapy,  in  hydrophobia,  202- 

205 
therapy,  in  influenza,  207 
therapv,    in   pneumonia,    234, 

225  " 
therapy,  in  small-pox,  235 
therapy,   in    streptococcus   in- 
fection. 242 
therapy,  in  tetanus,  266-268 
therapy,  in  typhoid  fever,  296, 
297 


Serum  •  therapv,    in    tuberculosis, 

287-289 
Shakespeare,    report    on    Fcri'iln's 
inoculations  against  cholera,  125- 
130 
Small-pox,  228-236 

after  vaccination,  230 
antitoxin  of  (?),  231-234 
protective  inoculations,  228 
second  attacks  of,  230 
Staphylococcus  infection,  immun- 
ity from,  244 
Sterilized  cultures,  immunitj'  pro- 
duced by,  40 
cultures,   in   treatment  of   ty- 
phoid fever,  297-299 
Sternberg's  explanation  of  acquired 

immunity,  41-45 
Streptococcus  infection,  241-244 
infection,      immunity     from, 
241-244 
Susceptibility,  race  differences,  4 

of  voung  animals,  4 
Swine  plague,  237-240 

plague,  protective  inoculatious 
in,  238 
Symptomatic  anthrax,  245-250    , 
anthrax,  preparation  of  "  vac- 
cines," 246,  248 
anthrax,     pr(>tective    inocula- 
tions in,  246-250 


Tetanus,  251-270 

antitoxin  of,  48,  258-2C3 
antitoxin,  dose  of,  261 
antitoxin,  in  milk,  53,  261-263 
antitoxin,    immiuiiziug   value 

of,  263 
bacillus,  cultivated  in  thymus 

bouillon,  261,  264 
bacillus,    toxic    products    of, 

253-256 
bacillus,  where  found,  252 
immunit}-,  47,  256-260 
immunity,  inherited,  266 
immunity,  in  horses,  265 
immunity,   Behring's   methbd 

of  i)roducing,  259,  260 
susceptible  animals,  2.")2 
serum-therapy  in,  266-268 
toxalbumin,  254,  255 
Thymus  bouillon,  38.  50.  261 
Toussainl's  method  of  attenuating 

virulence,  37,  82 
Toxins,  neutralization  of,  in  vitro, 
59 


INDEX. 


325 


Tuberculin  in  diagnosis  of  tuber- 
culosis, 286 

in  treatment  of   tuberculosis, 
283-286 

nielhods  of  preparing,  275-278 
Tuberculocidin,  Klebs's,'  277 
Tuberculosis,  271-289 

bacillus  of,  attenuated,  273 

bacillus  of,  biological  charac- 
ters, 272 

bacillus  of,  discovery,  271 

bacillus    of,    toxic    products, 
274 

bacillus  of,  where  found,  272 

immunity  from,  51,  281 

results  of  treatment  with   tu- 
Iterculin,  283-286 

serum-tlierapy  in,  287-289 

susceptibility  to,  3 
Tj'phoid   bacillus,  toxic   products 
of,  292-293 

bacillus,  where  found,  292 

fever,  292-300 


Tvphoid   fever,  serum-therapv  in, 

296-297 
fever,  treatment   bv  sterilized 

cultures,  297-299 
infection,      immunity     fiom, 

293-295 
infection  in  lower  animals,  8 
infection,  predisposing  causes, 

8 
Typhotoxin,  Brieger's,  292 

Vaccination,  discovery  of,  36 
necessity-  for  repeating,  231 
Vaccinia  in  the  cow.  229 
Vibrio     jMetchnikovi.     inimunitv 
from,  115 

Yellow  Fever,  301-320 

fever,  etioloiiy  not  determined, 

301 
fever  ''  germ  "  of  Freire.  802 
fever,   protective  inoculations 

in,  305-319 


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